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WO1996034091A1 - Procede permettant de mesurer des hormones dans des echantillons biologiques - Google Patents

Procede permettant de mesurer des hormones dans des echantillons biologiques Download PDF

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Publication number
WO1996034091A1
WO1996034091A1 PCT/US1996/000074 US9600074W WO9634091A1 WO 1996034091 A1 WO1996034091 A1 WO 1996034091A1 US 9600074 W US9600074 W US 9600074W WO 9634091 A1 WO9634091 A1 WO 9634091A1
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Prior art keywords
promoter
construct
gene
hormone
hydroxylase
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PCT/US1996/000074
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English (en)
Inventor
Hector F. Deluca
Nancy C. Arbour
Jean M. Prahl
Troy K. Ross
Claudia Zierold
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Wisconsin Alumni Research Foundation
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Application filed by Wisconsin Alumni Research Foundation filed Critical Wisconsin Alumni Research Foundation
Priority to EP96903342A priority Critical patent/EP0773989A1/fr
Priority to AU47459/96A priority patent/AU4745996A/en
Priority to JP8532474A priority patent/JPH11500626A/ja
Publication of WO1996034091A1 publication Critical patent/WO1996034091A1/fr

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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 present invention relates to methods for measuring hormone levels.
  • the field of the present invention is a method for measuring hormones, such as 1,25- dihydroxyvitamin D 3 , by use of a highly-sensitive reporter gene assay.
  • Biologically active 1,25-dihydroxyvitamin D 3 (1,25- (OH) 2 D 3 ) is an important mediator of bone and calcium homeostasis (H.F. DeLuca, e_t a_l. , Annu. Rev. Biochem. 52:411- 439, 1976).
  • circulating levels of l,25-(OH) 2 D 3 are utilized as a parameter of target organ vitamin D- mediated activity. Fluctuations in serum l,25-(OH) 2 D 3 concentrations are associated with abnormal l,25-(OH) 2 D 3 metabolism.
  • Several diseases in which aberrant plasma 1,25- (OH) 2 D 3 levels are observed include vitamin D-dependency rickets type I (H.
  • VDRE vitamin D responsive element
  • Hormone response elements have been reported for a variety of steroid, thyroid and retinoid hormones. See, for example, C. K. Glass, Endocrine Reviews 15[3]:391-406 and Wahli and Martinez, FASEB J. 5[2243]2249, 1991, for reviews. Lacking in the art is a sensitive and convenient method of quantitatively measuring hormones, such as l,25-(OH) 2 D 3 and vitamin D analogs.
  • the present invention is a method of quantitatively measuring hormone levels.
  • the method utilizes hormone response elements to measure steroid, retinoid and thyroid hormones.
  • the method comprises the steps of exposing a sample of a substance containing a hormone to a cell line transformed with a gene construct.
  • the gene construct comprises a promoter comprising a hormone response element operably connected to the reporter gene.
  • This hormone response element is responsive to the hormone that one wishes to measure.
  • the present invention is a method of quantitatively measuring l,25-(OH) 2 D 3 and vitamin D analogs .
  • the method comprises the step of exposing a sample of a substance containing l,25-(OH) 2 D 3 or an analog thereof to a cell line transformed with a gene construct.
  • the gene construct comprises a promoter containing a vitamin D response element operably connected to a reporter gene.
  • this reporter gene is not normally present in mammalian cells and is easily assayed.
  • the reporter gene is luciferase and the vitamin D response element is that found in the 25-hydroxyvitamin D 3 , 24- hydroxylase promoter.
  • the present invention is also a gene construct comprising a promoter operably connected to a DNA sequence encoding luciferase.
  • the promoter contains a vitamin D response element found in the 25-hydroxyvitamin D 3 , 24- hydroxylase ( "24-hydroxylase” ) promoter. (Nucleotides -1399 through +76 of the 24-hydroxylase gene are listed at SEQ ID NO:l.)
  • the promoter contains nucleotides -262 through -125 of the 24-hydroxylase promoter.
  • the promoter comprises nucleotides -1399 through +76 of the 24-hydroxylase gene.
  • the construct of claim 1 contains a hormone response element that is responsive to molecules selected from the group consisting of l,25-(OH) 2 D 3 and 25 hydroxy vitamin D analogs that are hydroxylated at position 1.
  • the hormone response element is responsive to 1,25-(OH) 2 D 3 .
  • the present invention is also a cell line transformed with the construct.
  • the cell line is permanently transformed. If one wishes to measure l,25(OH) 2 D 3 , the cell line is preferably either an osteosarcoma cell line or breast carcinoma cell line.
  • the present invention is also a kit for quantitatively measuring hormone levels.
  • the kit comprises a receptacle containing cells permanently transfected with a gene construc .
  • the gene construct comprises a promoter containing a hormone response element operably connected to a reporter gene.
  • the reporter gene is luciferase.
  • the kit also comprises a receptacle containing reagents necessary to analyze the reporter gene. For example, if the reporter gene were luciferase, the receptacle could contain luciferin substrate.
  • the gene construct described above is useful in a method of detecting the amount of l,25-(OH) 2 D 3 or analogs.
  • the method comprises exposing a sample of a substance containing 1 , 25-(OH) 2 D 3 or analog to the cell line transformed with the gene construct. One would then measure the amount of reporter gene luciferase expressed and compare this measurement to a standard curve.
  • One object of the present invention is to quantitatively measure hormone levels in hormone-containing samples, preferably biological samples. Another object of the present invention is to measure the amount of l,25-(OH) 2 D 3 in a biological sample, for example a patient's serum.
  • Another object of the present invention is to measure levels of 1, 25(OH) 2 D 3 at less than 10 nM.
  • Another object of the present invention is to measure levels of 1, 25-(OH) 2 D 3 less than 10 pM. Another object of the present invention is to measure levels of 1,25-(OH) 2 D 3 less than 1 pM.
  • the assay may use a variety of promoter constructs containing a vitamin D response element or elements .
  • Figs. IA and B diagram the construction of the rat 24- hydroxylase promoter/luciferase gene construct.
  • Fig. IA demonstrates a 1.5 kb Stu I fragment of the 24-hydroxylase gene promoter containing tandem VDREs that was sub-cloned into an expression vector pMAM Press ⁇ o -LUC.
  • Fig. IB demonstrates the orientation of the two recombinants p23 and plO.
  • Fig. 2 is a diagram of the two chimeric oligonucleotides (20- and 16-mers) which were synthesized to contain the 5' and 3' portions of luciferase gene and 24-hydroxylase promoter respectively.
  • Figs . 3A and B diagram the measurement of luciferase activity in transiently transfected cells.
  • Fig. 3A depicts T47-D cells
  • Fig. 3B depicts ROS 17/2.8 cells.
  • Figs. 4A and B diagram the expression of luciferase in stably transfected ROS 17/2.8 cells.
  • Fig. 4A depicts experiments with plasmid PIO
  • Fig. 4B depicts experiments with plasmid P23.
  • Fig. 5 diagrams the ratio of CAT activity of transfected cells dosed with 1,25-dihydroxy D (+D) over transfected cells dosed with vehicle (-D).
  • Fig. 6 diagrams CAT activities resulting from different combinations of half-sites of DRE 2 (-154 to -125).
  • Fig. 7 is a set of representative saturation curves (Figs. 7A, B, and C) with respective Scatchard plots (Figs. 7D, E, and F).
  • Figs. 7A and D describe experiments with -DREj (-262 to -238), Figs. 7B and E with -DRE 2 (-154 to -134) and Figs. 7C and F with nucleotides -260 to -136.
  • Fig. 8 diagrams binding properties and increased amounts of receptor in the presence of fixed amounts of probe.
  • Fig. 8A diagrams DRE- ⁇ and DRE 2 assayed separately, and
  • Fig. 8B diagrams DRE : and DRE 2 separated by their natural promoter sequence.
  • Fig. 9 is a standard curve for luciferase expression as a function of l,25(OH) 2 D 3 concentration. Description of the Invention 1. In general
  • the method involves the construction of a chimeric gene comprising a hormone response element operably placed upstream from a reporter gene. Preferably, more than one response element is included.
  • the response elements may be provided in their natural state as part of a hormone responsive promoter or may be operably attached to a promoter that is not responsive to hormone presence.
  • the method of the present invention is used to measure l,25-(OH) 2 D 3 or an analog.
  • the 24-hydroxylase promoter/luciferase gene reporter system of the present invention provides an alternative method for sensitive and accurate measurement of l,25-(OH) 2 D 3 and vitamin D analogs in biological samples.
  • the method we have developed is based on establishment of a cell line permanently-transfected with the gene construct described above.
  • the present invention begins with the preparation of a genetic construct comprising a reporter gene operably connected to a hormone response element.
  • Suitable response elements have been disclosed for many thyroid, retinoid and steroid hormones For example, Glass, 1994, supra reviews response elements for a large group of hormones and discusses the preferred placement of the core recognition motifs.
  • One of skill in the art could easily construct a suitable response element attached to the reporter gene of interest.
  • a gene encoding luciferase is operably connected to a promoter responsive to the presence of l,25(OH) 2 D 3 .
  • responsive we mean that the promoter is active only in the presence of the specific hormone.
  • operably connected we mean that the reporter gene is under control of the attached promoter or promoter plus response element.
  • VDREs vitamin D response elements
  • the 24- hydroxylase promoter described below is a preferred embodiment of a suitable promoter containing VDREs. (Preferably, nucleotides -1399 through +76 of the 24-hydroxylase gene are used. )
  • the 24-hydroxylase gene promoter is the most highly responsive control element identified thus far in a vitamin D target gene, due to low basal expression in the absence of l,25-(OH) 2 D 3 and highly inducible expression in the presence of the hormone.
  • the 24-hydroxylase promoter contains two vitamin D responsive elements which we term DRE X (at nucleotides -262 through -238) and DRE 2 (at nucleotides -154 through -125).
  • DRE X at nucleotides -262 through -2308
  • DRE 2 at nucleotides -154 through -125.
  • Fig. 5 and Example 2 demonstrate the placement of the naturally occurring half-sites in the 24-hydroxylase promoter and constructs designed to examine which of these half-sites are necessary for vitamin D-modulated transcription. The experiments in Example 1 were performed with the
  • Example 2 demonstrates that the thymidine kinase promoter is suitable for the present invention because the promoter has low basal activity. There are many other suitable and easily obtainable promoter sequences.
  • Example 2 and Fig. 5 demonstrate that a truncated version of the 24-hydroxylase promoter is also responsive to vitamin D and would be suitable for the present invention. Specifically, Example 2 and Fig. 5 demonstrate that a promoter construct containing half-sites b and a; half-sites d, c, and e; half-sites b, a, d, c, and e, and half-sites a, b, d, c, e separated by 93 bp are all suitable for the present invention. Constructs containing all 5 half-sites are preferred because the degree of enhancement of transcriptional activity is greater.
  • Half-sites b and a are included in nucleotides -262 through -238 of the 24- hydroxylase promoter.
  • Half-sites d, c, and e are included in nucleotides -154 through -125 of the 24-hydroxylase promoter.
  • Our experiments have shown us that it is the presence of the half-sites that are important in providing transcriptional enhancement.
  • the nucleotides between the half-sites may be varied with no change in activity. However, it is important to preserve a 3 nucleotide spacer between the half-sites. Note that it is not necessary that the naturally occurring 93 bp separate the two sets of half- sites .
  • Nuclear receptors also regulate the transcription of genes regulated by steroid hormones , thyroid hormones and retinoids . Each receptor protein is capable of binding to a specific DNA sequence upstream from particular genes that are targets for hormone regulation.
  • the protein-DNA interactions are mediated by a highly conserved DNA-binding domain that defines nuclear receptor super-family (C.K. Glass, et al . Endocrine Reviews 15 [ 3 ]: 391-407 , 1994). (This article is incorporated by reference as if fully set forth herein.)
  • the minimal target sequence recognized by the nuclear receptor DNA-binding domain consists of a 6 bp sequence and is sometimes referred to as a core recognition motif or a half-site.
  • Receptor proteins may be monomeric, heterodimeric or homodimeric.
  • Response elements that are to be recognized by monomeric receptor usually contain one copy of the core recognition motif.
  • response elements that are to be recognized by homodimer or heterodimer receptors usually have two recognition motifs with the characteristic spacing. Glass, et_ al. notes that "three features of a response element regulate the specificity of DNA recognition by a particular set of nuclear receptors: The precise sequence of the core recognition motif, the orientation of core recognition motifs with respect to each other..., and the spacing between core recognition motifs.”
  • the sequence AGAACA is preferentially recognized by the glucocorticoid, mineralocorticoid, progesterone, and androgen receptors.
  • the sequences AGGTCA and AGTTCA are preferentially recognized by the estrogen, thyroid hormone, retinoic acid and vitamin D receptor.
  • Steroid hormone response elements generally consist of a palindromic arrangement of the core recognition sequences while thyroid hormone, retinoic acid and vitamin D receptors may have direct repeats, palindromic or inverted palindromic arrangements of the core recognition sequence.
  • Monomeric receptors, such as NGFlb bind to response elements consisting of a single copy of the core recognition sequence (Glass, supra, 1994).
  • the estrogen response element has been characterized by Burch, et. al. , Mol . Cell . Biol . 8:1123-1131, 1933 and Seiler-Tuyns, et al., Nucl . Acid Res . 14:8755-8770, 1986.
  • Example 3 discloses the use of an estrogen response element in a construct using luciferase as a reporter gene. As described above, one would either use isolated response elements attached to a functioning non-hormone responsive promoter or a hormone-responsive promoter operably connected to a reporter gene.
  • a suitable promoter, as described above, is then operably connected to a reporter gene.
  • a reporter gene One of skill in the art will know of many different reporter genes which are suitable for the present invention.
  • the chloramphenicol acetyltransferase gene and the ⁇ -galactosidase gene are suitable for the present invention. All that is necessary is that one be able to measure the presence and amount of reporter gene expression.
  • the preferred reporter gene for the present invention is the luciferase gene.
  • the Examples below use the expression vector pMAM neo -LUC obtained from Clontech Laboratories Inc. in Palo Alto, CA as source of a luciferase gene-containing vector.
  • pGL2-basic, pGL2-enhancer, pGL2-promoter and pGL2 control plasmids available from Promega (Madison, WI) .
  • the genetic construct consisting of a VDRE-containing promoter and luciferase gene is typically placed in a vector for insertion into a cell line.
  • Suitable vectors include both plasmids and viral vectors.
  • the construct of the present invention has an opposite orientation to endogenous plasmid promoters, particularly if these endogenous plasmid promoters are hormone-responsive. The opposite orientation will prevent transcriptional read- through, false positives, and high backgrounds.
  • the present invention is also a cell line permanently transfected with a chimeric gene comprising a hormone response element operably connected upstream of a reporter gene. It is essential for the present invention that the cell line produce adequate levels of the particular hormone receptor corresponding to the hormone that is desired to be tested. For example, adequate levels of vitamin D receptor are required for l,25-(OH) 2 D 3 -enhanced reporter gene expression (H.M. Darwish, e_t ⁇ al. , Biochem. Biophys . Acta 1167:29-36, 1993; C.N. Hahn, et al., Nucleic Acids Res . 22:2410-2416, 1994).
  • a cell line that expresses vitamin D receptor is required for the present invention if vitamin D is to be assayed.
  • cell lines that naturally express the hormone receptor are especially preferred.
  • a cell line could be transformed to produce a recombinant version of a hormone receptor.
  • rat osteosarcoma cell line ROS 17/2.8
  • human breast carcinoma cell line T-47D
  • the human breast carcinoma cell line may be purchased from American Type Culture Collection.
  • the rat osteosarcoma cell line is available through Merck Sharp & Dohme Research Laboratories in Rahway, NJ.
  • cell lines can be used to measure l,25(OH) 2 D 3 .
  • the cell line must express vitamin D receptors, which may be measured in several ways.
  • U.S. patent no. 5,064,770 describes an assay for vitamin D receptor protein.
  • the cells are cultured and maintained by standard methods known to those of skill in the art. Similarly, the cells are transfected with the gene construct described above by standard methods.
  • the cell line is permanently transfected.
  • a cell line is "permanently" transfected if it maintains its transfected plasmid for more than 25 passages.
  • the cells are preferably grown in a neomycin-containing medium (e.g. GENETICIN* from GIBCO) . Cells that do not contain the plasmid will die in this environment. Thus, a permanently transfected cell line can be periodically "purified" by a pass or two through neomycin-containing medium. 4. Assay Method
  • the present invention is preferably a method of quantitatively determining a hormone level in a biological sample.
  • one will assay for l,25(OH) 2 D 3 .
  • the method is also suitable for determining 1,25- (OH) 2 D 3 analogs.
  • the analogs should have a 1 ⁇ -hydroxyl group and another hydroxyl in the region of the 24 - 27 carbons to be active, but can be otherwise modified.
  • the method of the present invention is suitable for determining both bound and free hormone levels.
  • the cells are incubated for 24 to 48 hours with the 1, 25-(OH) 2 D-containing sample.
  • cell lysates are then prepared, preferably by harvesting cells with 0.5% trypsin and 0.5 mM EDTA, washing twice with PBS, and then suspending in 100 ⁇ l of diluted luciferase lysis buffer as recommended by the supplier of the original luciferase-containing vector.
  • luciferase activity preferably a 20 ⁇ l aliquot is removed and mixed with 100 ⁇ l of luciferin substrate solution as described below in Example 1. Luciferase measurements should be initiated immediately after the addition of the substrate using a luminometer (Analytical Luminescence Laboratory, San Diego, CA) . The luminometer reading is compared to a standard curve prepared by exposure of the cell line with known amounts of hormone.
  • the method of the present invention is suitable for measuring l,25-(OH) 2 D 3 concentrations as low as 0.1 pM. Preferably, the l,25-(OH) 2 D 3 concentration is at least 1 pM.
  • kits The present invention is also a kit for quantitatively measuring hormone levels .
  • the kit comprises a receptacle containing a cell line permanently transfected with a gene construct.
  • the gene construct comprises a promoter containing a hormone response element operably connected to a reporter gene.
  • the reporter gene is luciferase.
  • the kit also comprises a receptacle containing reagents necessary to analyze the reporter gene. For example, if the reporter gene were luciferase, the receptacle could contain luciferin substrate.
  • the rat osteosarcoma cell line, ROS 17/2.8 was supplied by Dr. Gideon Rodan (Merck Sharp & Dohme Research Laboratories, Rahway, NJ). The cells were maintained in Ham's F-12 medium (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT). A human breast carcinoma cell line, T-47D, was purchased from American Type Culture Collection (Rockville, MD) and maintained in RPMI medium (GIBCO BRL) containing 5% serum and insulin at 1 unit/ml.
  • a 1.5 kb Stu I fragment of the rat 24-hydroxylase gene promoter was subcloned into the N ⁇ e I site of the expression vector, p AM n - Q -LUC (Clontech Laboratories, Inc., Palo Alto, CA) .
  • This vector contains a luciferase reporter gene and an SV40 promoter-driven neomycin resistance gene, which allows for selection of stably transfected cells.
  • an MMTV- LTR promoter controls dexamethasone-inducible luciferase expression.
  • Insertion of the segment into the multiple cloning site of pMAM- ⁇ c -LUC was performed such that luciferase expression was directed by the 24-hydroxylase promoter.
  • Putative recombinants were identified by hybridization with an oligonucleotide corresponding to the most 5' VDRE, 5 'CGCACCCGCTGAACC 3' (SEQ ID ⁇ 0:2), derived from the published sequence of the 24-hydroxylase gene (C. Zierold, et al . , supra, 1994).
  • Plasmid DNA was purified (Wizard Maxiprep kit, Promega, Madison, WI) and the orientation of the insert established by restriction enzyme digestion.
  • a recombinant with the 24-hydroxylase promoter-luciferase gene segment in the opposite orientation of the MMTV-LTR promoter was generated by digesting recombinant plasmid DNA with Sac I. The three fragments obtained were then re-ligated and colonies screened for the inverted segment by hybridization with the oligomer corresponding to the VDRE. Orientation of the 24-hydroxylase promoter was confirmed by restriction enzyme digestion and hybridization with two 24- chimeric oligonucleotides .
  • ROS 17/2.8 and T-47D cell lines were seeded in 6 cm dishes (approximately 2 X 10 5 cells per plate), grown overnight, and fed with serum-deficient medium. Transfections were performed using three constructs: (1) pMAM- ⁇ -LUC, the parental vector, (2) p240H/LUC #23, a recombinant with the 24-hydroxylase promoter in tandem with the MMTV-LTR promoter and (3) p240H/LUC #10, a recombinant with the 24-hydroxylase and MMTV-LTR promoters in opposite orientations.
  • Lysates were prepared by harvesting the cells with 0.5% trypsin in 0.5 M EDTA, washing twice with PBS, and suspending in 100 ⁇ l of diluted luciferase lysis buffer as recommended by the supplier (Promega). To measure luciferase activity in the samples, a 20 ⁇ l aliquot was removed and mixed with 100 ⁇ l of luciferin substrate solution (20 mM Tricine, 1 mM (MgC0 3 ) 4 Mg(OH) 2 , 2.7 mM MgSO A , 0.10 mM EDTA, 1 mM DTT, 270 ⁇ M Acetyl CoA, lithium salt, 470 ⁇ M D-Luciferin, and 526 ⁇ M ATP). Luciferase measurements were initiated immediately after addition of substrate using a luminometer (Analytical Luminescence Laboratory, San Diego, CA) .
  • T-47D cells The neomycin resistance gene allowed for selection of transformed cells using the aminoglycoside G418 (GIBCO BRL).
  • G418 sulfate 250 ⁇ g/ml
  • Fig. IA an expression vector, pMAM neo -LUC
  • the orientation of the insert in relation to the luciferase gene was determined by restriction enzyme digestion, based on the restriction maps of the 24- hydroxylase gene (GenBank data base accession no. U03112) and expression vector.
  • Fig. IB indicates the position and orientation of the MMTV-LTR and 24-hydroxylase promoters, which are juxtaposed near the 5' terminus of the luciferase gene.
  • the MMTV-LTR promoter which is glucocorticoid- inducible, controls luciferase activity endogenously.
  • the vector also harbors the ability to permit selection of stable transfectants that are antibiotic resistant.
  • a vector was constructed in which the 24-hydroxylase/luciferase gene segment was inverted with respect to the MMTV-LTR promoter. This manipulation was performed to eliminate low level, read-through transcription resulting from the endogenous promoter.
  • DNA from the recombinant plasmid was digested with Sac I, generating three fragments, 8.0, 2.9, and 0.8 kb, indicated in Fig. IB.
  • Digestion with Sac I and re-ligation of the three fragments produced a recombinant plasmid designated plO, in which the 24-hydroxylase promoter/luciferase gene, contained within the 2.9 kb segment, was inverted (Fig. IB).
  • Fig. IB Several putative recombinants that appeared to have inverted 24-hydroxylase promoter/luciferase gene segments based on restriction enzyme digestion were screened to confirm the presence of the inverted segment using two radiolabeled chimeric oligonucleotides (Fig.
  • oligomers were designed with the most 5' and 3' nucleotides derived from the luciferase gene and 24-hydroxylase promoter, respectively.
  • the 20-mer consisted of 7 nucleotides and the 16-mer was composed of 5 nucleotides from each segment, linked by a Sac I site.
  • oligonucleotides with differing lengths were chosen to ensure that only clones with both segments of the promoter/reporter gene would hybridize under standard stringency conditions .
  • clones with the 24-hydroxylase promoter-luciferase gene inserted in pMAM_, eo -LUC in the opposite direction with respect to the MMTV-LTR promoter gave a positive signal.
  • the original recombinant, plasmid p23 which contains the two promoters in tandem, did not hybridize significantly with either probe.
  • ROS 17/2.8 and T-47D cell lines were transiently transfected with the plasmids p23 and plO.
  • Fig. 3A shows that luciferase activity was increased approximately 10- to 15-fold upon the addition of 1 nM l,25-(OH) 2 D 3 to T-47D cells transfected with either recombinant plasmid. Background luciferase activity was higher in cells transfected with plasmid p23 than with plO. Approximately 5-fold stimulation of luciferase expression was observed in ROS 17/2.8 cells (Fig. 3B) . Background luciferase expression was higher in ROS 17/2.8 cells than in the T-47D cell line.
  • ROS 17/2.8 and T-47D cells were also transiently transfected with the parental vector, pMAM n - o -LUC, and subsequently treated with dexamethasone (100 nM) .
  • Treatment with dexamethasone increased luciferase expression only 2-fold, indicating that the MMTV-LTR promoter is not highly responsive to glucocorticoid treatment in either of these cell types.
  • ROS 17/2.8 cells were permanently transfected with plasmids plO and p23, which have the 24-hydroxylase promoter/luciferase gene segment in either orientation.
  • ROS 17/2.8 cells were grown to ⁇ 75% confluency in F-12 medium supplemented with 10% FBS. The cells were harvested and diluted to 1-5 x 10 3 cells in 200 ⁇ L of F-12 medium containing 10% FBS in a 96 well plate (Falcon No. 3872). The cells were then incubated over- night at 37°C, 5% C0 2 , 95% humidity.
  • the l,25(OH) 2 D 3 standards in 2 ⁇ L of 100% ethanol were added to 50 ⁇ L of F-12, 10% FBS medium. This mixture was added to each well containing transformed cells. All samples were analyzed in triplicate. The cells were incubated 18 hours at 37°C, 5% C0 2 , 95% humidity. The cells were then washed 2 times with PBS, pH 7.2, and the plate blotted with a paper towel .
  • the cells were then lysed by the addition of 100 ⁇ L lysis buffer.
  • the plate was shaken on ice for 1 hour for complete lysis.
  • the lysis buffer was: 25 mM Tris- phosphate, pH 7.8; 2 mM dithiothreitol (DTT); 2 mM 1,25- diaminocyclohexane-N,N,N' ,N'-tetraacetic acid (CDTA); 10% glycerol; 1% Triton-X-100.
  • Lysis buffer was mixed with the lysed cells using a pipette.
  • the relative light units were read for 10 seconds on a Monolight 2010 luminometer.
  • the substrate was automatically dispensed by the luminometer.
  • the assay was done in duplicate or triplicate. Results are described in Table 2 and Fig. 9.
  • oligonucleotides were generated by DNA synthesizer (ABI, La Jolla, CA) as shown below. These oligonucleotides were designed to have Xba I termini for labeling and subcloning. Table 3 describes the different oligonucleotides. The larger oligonucleotides (over 100 bp) were amplified by PCR using the primers listed at SEQ ID NOs:10, 11, 12, and 13. The 24-hydroxylase promoter region was used as template. TABLE 3
  • Chicken 24-hydroxylase cDNA was used as template for the nonspecific DNA (Ismail and DeLuca, unpublished results), and the DRE sequences were included in the primers as shown above.
  • the recombinant reporter gene constructs were obtained by cloning the oligonucleotides mentioned above into the Xbal site of the pBLCAT2 vector (Luckow and Schuetz, Nucl . Acids Res. 15:5490, 1987). 2.5 ⁇ g per 60 mm dish of these constructs were transfected into NRK-52E rat kidney cells using the LIPOFECTIN ® method (GIBCO/BRL, Gaithersburg, MD) . The LIPOFECTIN ® :DNA ratio was 3:1. Transfected cells were dosed with l,25-(OH) 2 D 3 to a final concentration of 10 nM and incubated for 20-24 hours.
  • the cells were then harvested, and the activity of the chloramphenicol acetyl transferase (CAT) enzyme was measured as described before (Zierold, et. al. , Proc. Natl. Acad. Sci. USA 91:900-902, 1994) . Determination of Dissociation Constant (K,*).
  • CAT chloramphenicol acetyl transferase
  • the probe obtained by PCR using chicken 24-hydroxylase cDNA was endlabeled utilizing ⁇ [ 32 P]dATP (DuPont/NEN) and the T4 polynucleotide kinase (Promega, Madison, WI) .
  • the labeled probes were purified by polyacrylamide gel electrophoresis followed by electroelution. The final probe concentrations after purification were 0.5 ng/ ⁇ l or 1 ng/ ⁇ l. Binding of the VDR to the labeled DNA probes was performed as described previously (Darwish and DeLuca, Proc. Natl. Acad. Sci .
  • porcine intestinal nuclear extract containing VDR and the labeled DNA were incubated at 4° C for 2 hours, the time needed for maximal binding.
  • the final KC1 concentration was 75 mM, the concentration at which maximal binding occurs in the absence of 1, 25-(OH) 2 D 3 .
  • 1, 25-(OH) 2 D 3 was added to the reaction, determinations were made at both 75 mM and 150 mM KCl, the latter being the concentration at which maximal binding occurs in the presence of 1, 25-(OH) 2 D 3 .
  • the VDR-DNA complexes and unbound DNA were quantitated from the dried gels using the Betascope Analyzer (Betagen, Waltham, MA) . All affinity measurements were calculated using computer assisted graphics.
  • Fig . 5 shows the structure of each pBLCAT2 construct used and the respective reporter gene activity induced by 1 , 25-(OH) 2 D 3 .
  • each construct contains the thymidine kinase (tk) promoter and one or two DREs, drawn as arrows and labeled with letters representing each half- site. The direction of the arrows indicates that the consensus sequence resides on the antisense strand.
  • DRE :
  • DRE 2 (e...c...d) is located between -154 and -125 upstream of the transcription start site.
  • the sequence of each DRE is indicated.
  • the 93 bp that separate the two DREs represent the separation in the natural promoter. For comparative reasons, the activity of the construct containing its own promoter and 1400 bp upstream was also included (last line).
  • the entire DRE unit including 5 ' half-sites produced a dramatic stimulation of CAT activity. Since this includes the DRE at -262 and the three half-sites at -154, the contribution of each was examined.
  • the DRE at -154 is not responsive to l,25-(OH) 2 D 3 by itself but requires the presence of a third half-site located at the 3 '-end. All three sites are required for
  • Fig. 6 diagrams CAT activities resulting from different combinations of half-sites of DRE 2 (-154 to -125). Referring to Fig. 6, the small letters correspond to the half-sites depicted in Fig. 5. Activities are expressed as total counts of acetylated chloramphenicol obtained in 19 hours on the Betascope analyzer. All reactions contained 100 ⁇ g protein. The CAT assays were carried out as explained in Materials and Methods . In the intact promoter, a 93 bp segment separates the two DREs. The experiments diagrammed in Fig. 5 demonstrate that when this 93 bp segment is deleted, the transactivation by l,25-(OH) 2 D 3 is unchanged.
  • the values represent means ⁇ S. D.
  • the numbers refer to their location upstream of the transcriptional start-site in the 24-hydroxylase gene. Arrows represent half-sites and their orientation indicates that the consensus sequence is found on the antisense strand. Determinations were carried out as explained in Materials and Methods .
  • Fig. 7 diagrams representative saturation curves (Figs. 7A, B, and C) with respective Scatchard plots (Figs. 7D, E, and F).
  • Figs. 7A and D represent DRE*. (-262 to -238)
  • Figs. 7B and E represent DRE 2 (-154 to -134)
  • Figs. 7C and F represent nucleotides -260 to -136.
  • Average K d s (-1/slope) for several determinations are shown in Table 4.
  • Table 4 presents the K d S determined for the DREs by themselves, their half-sites and the entire D-responsive region of the 24-hydroxylase promoter. Data points were obtained from gel retardation assays described in Materials and Methods . The amount of complex formed was plotted versus the amount of unbound probe as shown in Figs. 7A, B, and C. These saturation plots show that the DREs separately (Figs. 7A and 7B) reach saturation at a 250-fold higher concentration than both together.
  • the dissociation constants (K d ) were calculated from Scatchard analysis as shown in the accompanying plots in Figs. 7D, E, and F (Scatchard, G., Ann. N.Y. Acad. Sci. 51:660-672, 1949). All Scatchard plots in Fig. 7 show biphasic binding. Two K d s were calculated for each fragment.
  • Fig. 8 diagrams the binding properties of increasing amounts of receptor in the presence of fixed amounts o_ probe.
  • Fig. 8A diagrams DREj and DRE 2 assayed separately, and
  • Fig. 8B diagrams DREj and DRE 2 separated by their natural promoter sequence.
  • the promoter region of the rat 1 , 25-(OH) 2 D-24-hydroxylase gene contains the most powerful vitamin D-responsive element system reported to date.
  • This system includes two distantly separated response elements, one of which contains two half-sites separated by a 3 nonspecific bases in accordance with K. Umesono, et al. , Cell 65:1255-1266 (1991), and the other response element contains 3 half-sites that are essential for the transactivation activity by l,25-(OH) 2 D 3 and its receptor.
  • the DRE site closest to the transcriptional start site contains 3 half-sites, each separated by 3 bp, which is an unusual arrangement for vitamin D responsive genes.
  • vitellogenin gene has two EREs (J. Burch, e_t al. , Mol.
  • mice mammary tumor virus (MMTV) long terminal repeat contains multiple copies of the GRE (C. Scheidereit, et al., Nature 304:749-752, 1983).
  • the results are not entirely comparable to the data obtained using a reporter gene system containing the thymidine kinase (tk) promoter used to analyze fragments and constructs.
  • tk thymidine kinase
  • the 24-hydroxylase promoter in the promoterless CAT reporter system shows low basal activity which accounts for the higher ratio of +D/-D response.
  • the first approach uses a response element found at -356 to -331 of the vitellogenin promoter.
  • the second approach uses sequences at nucleotides -630 to -331 and -1133 to -331 of the same promoter.
  • oligonucleotides were as follows: 5' CTAGAGGTCAACATAACCTGGGCAAAACCA
  • TK TK promoter in pBLCAT2.
  • MOLECULE TYPE DNA (genomic)
  • ATGTAAACAC TAAAATAAAT TATTAATAAA TAAGAAGCAT TGGCGTGAAA TTCCTGGTTT 300
  • CTGTGATAAC CCTGCCTGCT TTAGGTGGGC TTTAGGGAGA AAGTGGGGCT CTTGGGAACC 900
  • CTGTCCTCAG GGACCTTGCC CGCCCTGCAT GGCGATTGTG CAAGCGCACG TTTGGGCTCC 1440
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:2: CGCACCCGCT GAACC 15
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:3: GGGTACCGAG CTCTGTTCTA 20
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:5: CTAGAGAGCG CACCCGCTGA ACCCTGGGCT 30
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:6: CTAGACGGCG CCCTCACTCA CCTCGCT 27
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:7: CTAGACGGCG CCCTCACTCA CCTCGCTGAC TCCAT 35
  • MOLECULE TYPE oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO:9: CTAGAGAGCT CTGAACCCTG GGCT 24
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:10: CAGGCGTTCT AGAGCGCACC CGCTGAACCC 30
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:11: CAGGCGTTCT AGAGAGGTGA GTGAGGGCGC 30 (2) INFORMATION FOR SEQ ID NO:12:
  • MOLECULE TYPE oligonucleotide
  • oligonucleotide SEQUENCE DESCRIPTION: SEQ ID NO: 12: GAGCGCACCC GCTGAACCTC TAGAGGTCAG CTTTCCAAGA AG 42
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:13: GCGAGGTGAG TGAGGGCGTC TAGAAGCAAA CTGTTGGCCG TC 42
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:14: CTAGAGGTCAACATAACCTGGGCAAAACCA 30
  • MOLECULE TYPE Other Nucleic Acid
  • SEQUENCE DESCRIPTION SEQ ID NO:16: GGGTGAATGA GGACA 15
  • MOLECULE TYPE Other Nucleic Acid
  • SEQUENCE DESCRIPTION SEQ ID NO:17: GGGTGAACGG GGGCA 15
  • MOLECULE TYPE Other Nucleic Acid
  • SEQUENCE DESCRIPTION SEQ ID NO:18: GGTTCACGAG GTTCA 15 (2) INFORMATION FOR SEQ ID NO: 19:
  • MOLECULE TYPE Other Nucleic Acid
  • SEQUENCE DESCRIPTION SEQ ID NO:19: GGTTCAGCGG GTGCG 15
  • MOLECULE TYPE Other Nucleic Acid
  • SEQUENCE DESCRIPTION SEQ ID NO:20: GGGGGATGTG AGGAG 15

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Abstract

Produit de recombinaison génique, lignée cellulaire et procédé destinés à la mesure d'hormones, de préférence la 1,25-dihydroxyvitamine D3 et des analogues de vitamine D. Le produit de recombinaison génique comprend un promoteur relié de manière active à un gène marqueur, ce promoteur contenant un élément sensible à une hormone. La lignée cellulaire est transformée de manière irréversible au moyen du produit de recombinaison, et le procédé consiste à exposer un échantillon de la substance contenant l'hormone à la lignée cellulaire, à mesurer le niveau d'expression du gène marqueur et à établir une corrélation entre ce niveau d'expression et la concentration d'hormone.
PCT/US1996/000074 1995-04-28 1996-01-11 Procede permettant de mesurer des hormones dans des echantillons biologiques WO1996034091A1 (fr)

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EP96903342A EP0773989A1 (fr) 1995-04-28 1996-01-11 Procede permettant de mesurer des hormones dans des echantillons biologiques
AU47459/96A AU4745996A (en) 1995-04-28 1996-01-11 Method for measuring hormones in biological samples
JP8532474A JPH11500626A (ja) 1995-04-28 1996-01-11 生物学的試料中のホルモン類の測定方法

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DE19855013A1 (de) * 1998-11-20 2000-07-20 Schering Ag Androgen- und Progesteron-Rezeptor bindendes Hormon Response Element
EP0992189A4 (fr) * 1997-04-30 2000-12-27 Takeda Chemical Industries Ltd Animaux porteurs d'un gene transfere de 25-hydroxyvitamine d3-24-hydroxylase

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EP0325849A2 (fr) * 1987-12-02 1989-08-02 The Salk Institute For Biological Studies Composition de récepteur de l'acide rétinoique et procédé d'identification de ligands

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EP0325849A2 (fr) * 1987-12-02 1989-08-02 The Salk Institute For Biological Studies Composition de récepteur de l'acide rétinoique et procédé d'identification de ligands
EP0540065A1 (fr) * 1987-12-02 1993-05-05 The Salk Institute For Biological Studies Composition du récepteur de l'acide rétinoique

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C.K. GLASS: "Differential recognition of target genes by nuclear receptor monomers, dimers, and heterodimers", ENDOCRINE REVIEWS, vol. 15, no. 3, June 1994 (1994-06-01), TH EENDOCRINE SOCIETY, US, pages 391 - 407, XP000572339 *
CHEN, KAI-SHUN ET AL: "Cloning of the human 1.alpha.,25-dihydroxyvitamin D-3 24-hydroxylase gene promoter and identification of two vitamin D - responsive elements", BIOCHIM. BIOPHYS. ACTA (1995), 1263(1), 1-9 CODEN: BBACAQ;ISSN: 0006-3002, 25 July 1995 (1995-07-25), XP000572343 *
H.M. DARWISH ET AL.: "Phosphorylation is involved in the transcriptional activation by the 1,25-dihydroxyvitamin D3 receptor", BIOCHIMICA BIOPHYSICA ACTA, vol. 1167, no. 1, 17 March 1993 (1993-03-17), ELSEVIER SCIENCE, AMSTERDAM, NL, pages 29 - 36, XP000572602 *
KEPHART, DANIEL D. ET AL: "Retinoid X receptor isotype identity directs human vitamin D receptor heterodimer transactivation for the 24-hydroxylase vitamin D response elements in yeast", MOL. ENDOCRINOL. (1996), 10(4), 408-19 CODEN: MOENEN;ISSN: 0888-8809, April 1996 (1996-04-01), XP000572335 *
Y. OHYAMA ET AL.: "Identification of a vitamin D-responsive element in the 5'-flanking region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene", J. BIOL. CHEM., vol. 269, no. 14, 8 April 1994 (1994-04-08), AM. SOC. BIOCHEM. MOL.BIOL.,INC.,BALTIMORE,US, pages 10545 - 10550, XP002005153 *
ZIEROLD, CLAUDIA ET AL: "Two vitamin D response elements function in the rat 1,25-dihydroxyvitamin D 24 - hydroxylase promoter", J. BIOL. CHEM. (1995), 270(4), 1675-8 CODEN: JBCHA3;ISSN: 0021-9258, 1995, XP002005154 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0992189A4 (fr) * 1997-04-30 2000-12-27 Takeda Chemical Industries Ltd Animaux porteurs d'un gene transfere de 25-hydroxyvitamine d3-24-hydroxylase
US6316689B1 (en) 1997-04-30 2001-11-13 Takeda Chemical Industries, Inc. 25-hydroxyvitamin D3 24-hydroylase transgenic rats
DE19855013A1 (de) * 1998-11-20 2000-07-20 Schering Ag Androgen- und Progesteron-Rezeptor bindendes Hormon Response Element
DE19855013C2 (de) * 1998-11-20 2001-09-13 Schering Ag Androgen- und Progesteron-Rezeptor bindendes Hormon Response Element
US6518041B1 (en) 1998-11-20 2003-02-11 Schering Aktiengesellschaft Hormone response element that binds an androgen and progesterone receptor

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