WO1996034091A1

WO1996034091A1 - Method for measuring hormones in biological samples

Info

Publication number: WO1996034091A1
Application number: PCT/US1996/000074
Authority: WO
Inventors: Hector F. Deluca; Nancy C. Arbour; Jean M. Prahl; Troy K. Ross; Claudia Zierold
Original assignee: Wisconsin Alumni Research Foundation
Priority date: 1995-04-28
Filing date: 1996-01-11
Publication date: 1996-10-31
Also published as: EP0773989A1; JPH11500626A; AU4745996A

Abstract

A gene construct, cell line, and method useful for measuring hormones, preferably 1,25-dihydroxyvitamin D3 and vitamin D analogs is disclosed. The gene construct comprises a promoter operably connected to a reporter gene, wherein the promoter contains a hormone response element. The cell line is permanently transformed with the construct. The method comprises exposing a sample of the substance containing the hormone to the cell line, measuring the expression level of the reporter gene and correlating the expression level with hormone concentration.

Description

METHOD FOR MEASURING HORMONES IN BIOLOGICAL SAMPLES

Field of the Invention

The present invention relates to methods for measuring hormone levels. Specifically, the field of the present invention is a method for measuring hormones, such as 1,25- dihydroxyvitamin D₃, by use of a highly-sensitive reporter gene assay.

Background Measurement of 1 , 25-dihydroxyvitamin D*-.

Biologically active 1,25-dihydroxyvitamin D₃ (1,25- (OH)₂D₃) is an important mediator of bone and calcium homeostasis (H.F. DeLuca, e_t a_l. , Annu. Rev. Biochem. 52:411- 439, 1976). Clinically, circulating levels of l,25-(OH)₂D₃ are utilized as a parameter of target organ vitamin D- mediated activity. Fluctuations in serum l,25-(OH)₂D₃ concentrations are associated with abnormal l,25-(OH)₂D₃ metabolism. Several diseases in which aberrant plasma 1,25- (OH)₂D₃ levels are observed include vitamin D-dependency rickets type I (H. Koyama, e_t a . , Anal. Biochem. 205:213- 219, 1992), primary hyperparathyroidism (A.E. Broadus, e_t al.., N. Enql. J. Med. 302:421-426, 1980), and chronic kidney failure (D.A. Feinfeld, et. a . , Kidney Int. 33:1049-1053, 1988). Decreased serum l,25-(OH)₂D₃ levels have also been correlated with symptomatic immunodeficiency viral infections (C. Haug, et al., J^ Inf. Pis. 169:889-892, 1994). Measurement of serum l,25-(OH)₂D₃ levels has relied on extensive HPLC purification (J.A. Eisman, e_t al. , Arch. Biochem. Biophys . 176:235-243, 1976; P.H. Stern, et al. , Anal. Biochem. 102:22-30), competitive binding assays (J.A. Eisman, et a_l. , Science 193:1021-1023, 1976), or the availability of antibodies against l,25(OH)₂D₃ (R.E.A.

Bouillon, Clin. Chem. 26:562-567, 1980; E.B. Maywer, Clin. Che . Acta 190:199-204, 1990). These procedures have been useful, but limited by their inability to detect low quantities (pM levels) of circulating l,25-(OH)₂D₃ with ease and accuracy. In addition, clinical trials with vitamin D analogs for the treatment of osteoporosis require a convenient assay for analysis of their serum levels, which is often difficult with the currently available methods. l,25-(OH)₂D₃ has been shown to stimulate 25- hydroxyvitamin D₃, 24-hydroxylase expression. (K.-S. Chen, et al., Proc. Natl. Acad. Sci . USA 90:4543-4547, 1993; C. Zierold, et al.. Proc. Natl. Acad. Sci. USA 91:900-902, 1994). This expression pattern is presumably due to the presence of tandem vitamin D responsive element (VDRE) sequences, separated by 93 nucleotides and positioned within the first 262 nucleotides upstream of the transcriptional start site (Zierold, e_t a_l. , supra, 1994). These two VDRE sequences are located between nucleotides -262 to -238 and - 154 to -134 (C. Zierold, et. a . , supra, 1994; Y. Ohyama, et. al. , Biochemistry 32:76-82, 1993). Recently, Zierold, et al. (supra, 1994) demonstrated that in transiently transfected rat kidney NRK cells, homologous and heterologous 24- hydroxylase promoters induced a 4- to 10-fold increase in chloramphenicol acetyltransferase (CAT) activity upon treatment of the cells with 10 nM 1,25-(OH)₂D₃.

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)₂D₃ and vitamin D analogs.

Summary of the Invention In one embodiment, the present invention is a method of quantitatively measuring hormone levels. Preferably, 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.

In another embodiment, the present invention is a method of quantitatively measuring l,25-(OH)₂D₃ and vitamin D analogs . The method comprises the step of exposing a sample of a substance containing l,25-(OH)₂D₃ 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. Preferably, this reporter gene is not normally present in mammalian cells and is easily assayed. Preferably, the reporter gene is luciferase and the vitamin D response element is that found in the 25-hydroxyvitamin D₃, 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 D₃, 24- hydroxylase ( "24-hydroxylase" ) promoter. (Nucleotides -1399 through +76 of the 24-hydroxylase gene are listed at SEQ ID NO:l.) Preferably, the promoter contains nucleotides -262 through -125 of the 24-hydroxylase promoter. Most preferably, the promoter comprises nucleotides -1399 through +76 of the 24-hydroxylase gene. Most preferably, the construct of claim 1 contains a hormone response element that is responsive to molecules selected from the group consisting of l,25-(OH)₂D₃ and 25 hydroxy vitamin D analogs that are hydroxylated at position 1. Most preferably, the hormone response element is responsive to 1,25-(OH)₂D₃.

The present invention is also a cell line transformed with the construct. Preferably, the cell line is permanently transformed. If one wishes to measure l,25(OH)₂D₃, 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. Preferably, 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)₂D₃ or analogs. The method comprises exposing a sample of a substance containing 1 , 25-(OH)₂D₃ 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)₂D₃ in a biological sample, for example a patient's serum.

Another object of the present invention is to measure levels of 1, 25(OH)₂D₃ at less than 10 nM.

Another object of the present invention is to measure levels of 1, 25-(OH)₂D₃ less than 10 pM. Another object of the present invention is to measure levels of 1,25-(OH)₂D₃ less than 1 pM.

It is a feature of the present invention that the assay is quick and convenient.

It is another feature of the present invention that the assay may use a variety of promoter constructs containing a vitamin D response element or elements .

It is another feature of the present invention that the assay construct results in a very low background level of reporter gene expression. Other objects, features and advantages of the present invention will become apparent after examination of the specification, claims and drawings. Description of the Drawings 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„_βo-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, and 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, and 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 DRE₂ (-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 -DRE₂ (-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 DRE₂ assayed separately, and Fig. 8B diagrams DRE_: and DRE₂ separated by their natural promoter sequence.

Fig. 9 is a standard curve for luciferase expression as a function of l,25(OH)₂D₃ concentration. Description of the Invention 1. In general

As a more sensitive and widely applicable method, we have developed a molecular approach for the detection and measurement of hormones, preferably l,25-(OH)₂D₃ and vitamin D analogs. 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.

Preferably, the method of the present invention is used to measure l,25-(OH)₂D₃ 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)₂D₃ 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.

2. Gene Constructs

A. Suitable Promoters

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. Preferably, a gene encoding luciferase is operably connected to a promoter responsive to the presence of l,25(OH)₂D₃. By "responsive" we mean that the promoter is active only in the presence of the specific hormone. By "operably connected" we mean that the reporter gene is under control of the attached promoter or promoter plus response element.

For example, one could use vitamin D response elements (VDREs) to create a vitamin-D responsive promoter. 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)₂D₃ 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 DRE₂ (at nucleotides -154 through -125). We describe these vitamin D response elements in terms of "half-sites" , so named because more than one of these sites is required to make a complete vitamin D response element. Three half-sites separated by 3 bp are needed in DRE₂ while two half-sites separated by 3 bp are needed for DRE_j . 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

24-hydroxylase promoter. The experiments in Example 2 were performed with vitamin D responsive elements (taken from the 24-hydroxylase promoter) upstream from a non-vitamin D response promoter. Our experiments have shown that these vitamin D response elements are sufficient to cause vitamin D-modulated transcriptional activity when placed upstream of a functioning promoter. Therefore, either combination of promoter and response element is suitable for the present invention. When we refer to a promoter "containing" a hormone response element, we mean that either the response element may be attached upstream of a functioning promoter or may be part of a functional promoter. The "promoter" then encompasses both the original functioning promoter and the response element construction.

Example 2 below 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 .

Transcription is enhanced when both sets of VDREs are available. However, an increase of expression when only one VDRE is present (either DRE-_, or DRE₂) is suitable for the present invention. Our examples are performed with a 24- hydroxylase gene isolated from rat. We envision that similar vitamin D response elements will be found in 24-hydroxylase promoters from other species as well. One of skill in the art would perform an analysis similar to that performed in Experiment 2 to determine which sequences are necessary for the present invention. Other known vitamin D response elements are also suitable for the present invention because they allow increased gene expression in the presence of vitamin D. Table 1 lists these elements.

TABLE 1

Vitamin D Response Elements

Gene Nucleotide sequence Reference

Rat calbindin-D_9k GGGTGT CGG AAGCCC Darwish and DeLuca, Proc. Natl. Acad. Sci. U.S.A. 89:603-607 (1992) (SEQ ID NO: 15)

Rat osteocalcin GGGTGA ATG AGGACA Demay, et al., Proc. Natl. Acad. Sci. U.S.A. 87:369- 373 (1990) (SEQ ID N0:16) Human osteocalcin GGGTGA ACG GGGGCA Ozono, et al., J. Biol. Chem. 265:21881-21888 (1990) (SEQ ID N0:17)

Mouse osteopontin GGTTCA CGA GGTTCA Noda, et al. , Proc. Natl. Acad. Sci. U.S.A. 87:9995- 9999 (1990) (SEQ ID N0:18)

Rat 25-hydroxy- GGTTCA GCG GGTGCG Zierold, et al., Proc. vitamin D Natl. Acad. Sci. U.S.A. 2 -hydroxylase 91:900-902 (1993) (SEQ ID NO: 19) Mouse calbindin-D_28k GGGGGA TGT GAGGAG Gill and Christakos, Proc. Natl. Acad. Sci. U.S.A. 90:2984-2988 (1993) (SEQ ID NO: 20)

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. In contrast, 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."

Two general classes of core recognition motifs that confer positive transcriptional responses have been reported. 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).

Therefore, if one wished to assay a hormone other than vitamin D, one would use a response element suitable for that hormone. For example, 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 below 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. B. Construction of a Reporter Gene-Containing Vector A suitable promoter, as described above, is then operably connected to 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 β-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. Other sources of a functional luciferase gene are the 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.

If a plasmid vector is selected, one must address the problem of endogenous plasmid promoters. Preferably, 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.

3. Cell Lines

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)₂D₃-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). Therefore, a cell line that expresses vitamin D receptor is required for the present invention if vitamin D is to be assayed. Especially preferred are cell lines that naturally express the hormone receptor. However, a cell line could be transformed to produce a recombinant version of a hormone receptor.

If one wishes to assay for vitamin D, the Examples below demonstrate that rat osteosarcoma cell line (ROS 17/2.8) and human breast carcinoma cell line (T-47D) are suitable for the present invention because of their high vitamin D receptor content (S.C. Manolagas, et_. aJL. , J_;_ Biol. Chem. 255:4415- 4417, 1980), consistent with target sites for vitamin D action. 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.

We envision that other cell lines can be used to measure l,25(OH)₂D₃. The cell line must express vitamin D receptors, which may be measured in several ways. For example, 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.

Preferably, 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. Preferably, one will assay for l,25(OH)₂D₃. However, the method is also suitable for determining 1,25- (OH)₂D₃ analogs. The analogs should have a 1 α-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. One could chemically treat the biological sample, for example with a suitable organic solvent or with high temperatures, and liberate the bound hormone from the protein receptor. After such a sample preparation, one would then be able to assay for the total amount of hormone in a sample. Alternatively, if one did not liberate the bound hormone, one would be measuring the free hormone concentration. Either measurement might be appropriate for a particular use.

One first obtains a biological sample and then incubates the cell line described above with various dilutions of the sample. The cells are then assayed for expression of the reporter gene. The expression level can be compared to a standard curve to determine free hormone concentration in the sample. If one wishes to test for l,25(OH)₂D₃, the dichloromethane extraction/Sep-Pak Silica chromatography method described by Koyama, et al. (supra., 1992) for purification of vitamin D compounds may be useful prior to testing in the present assay. The authors indicate that this procedure eliminates 25-OH-D₃, which is the major form of vitamin D present in serum that may compete with 1,25- (OH)₂D₃.

Preferably, the cells are incubated for 24 to 48 hours with the 1, 25-(OH)₂D-containing sample. Preferably, 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 μl of diluted luciferase lysis buffer as recommended by the supplier of the original luciferase-containing vector.

In an alternative to preparing cell lysates, one may leave cells attached to a well, and test the supernatant after the addition of lysis buffer, as described below in Example 3.

One would then determine the level of reporter gene expression. To measure luciferase activity in the samples, preferably a 20 μl aliquot is removed and mixed with 100 μl 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)₂D₃ concentrations as low as 0.1 pM. Preferably, the l,25-(OH)₂D₃ concentration is at least 1 pM.

5. 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. Preferably, 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.

Examples

Example 1-Characterization of a reporter gene assay for 1,25-

(OH)₇D₃

A. Materials and Methods

Cell culture 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.

Construction of reporter gene construct

A 1.5 kb Stu I fragment of the rat 24-hydroxylase gene promoter, from -1399 to +76 nucleotides of the published sequence (C. Zierold, et. al. , supra, 1994 and SEQ ID N0:1), was subcloned into the NΛe I site of the expression vector, p AM_n-_Q-LUC (Clontech Laboratories, Inc., Palo Alto, CA) . I This vector contains a luciferase reporter gene and an SV40 promoter-driven neomycin resistance gene, which allows for selection of stably transfected cells. In addition, 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 Ν0:2), 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 . Transient cell transfection

ROS 17/2.8 and T-47D cell lines were seeded in 6 cm dishes (approximately 2 X 10⁵ 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. 2 μg of p240H/LUC plasmid DNA were introduced into the cells using LIPOFECTIN^® reagent as directed by the manufacturer (GIBCO BRL) the cells were incubated at 37°C overnight, washed, and fed with serum-sufficient medium. l,25-(OH)₂D₃ (purchased from Tetronics, Inc., Madison, WI) was added at a final concentration of 1 nM and the cells incubated an additional 24 to 48 hours. Some plates received ethanol alone or 100 nM dexamethasone (Sigma Chemical Co., St. Louis, MO) to measure expression from the MMTV-LTR promoter.

Luciferase assay

Lysates were prepared by harvesting the cells with 0.5% trypsin in 0.5 M EDTA, washing twice with PBS, and suspending in 100 μl of diluted luciferase lysis buffer as recommended by the supplier (Promega). To measure luciferase activity in the samples, a 20 μl aliquot was removed and mixed with 100 μl of luciferin substrate solution (20 mM Tricine, 1 mM (MgC0₃)₄Mg(OH)₂, 2.7 mM MgSO_A, 0.10 mM EDTA, 1 mM DTT, 270 μM Acetyl CoA, lithium salt, 470 μM D-Luciferin, and 526 μM ATP). Luciferase measurements were initiated immediately after addition of substrate using a luminometer (Analytical Luminescence Laboratory, San Diego, CA) .

Stable cell transfections

The recombinant plasmid with the inverted 24- hydroxylase/luciferase segment, designated plO, was stably transfected essentially as described by F.M. Ausubel, et al. , in Current Protocols in Molecular Biology, Vol. 1, 1987 in T- 17

47D cells. The neomycin resistance gene allowed for selection of transformed cells using the aminoglycoside G418 (GIBCO BRL). T-47D cells at a density of 2 X 10⁵ cells per plate were seeded in T-25 flasks in complete medium and upon reaching confluence, were split 1:15. Cells were washed, serum-deficient medium added, and the cells transfected with 2 μg of DNA using LIPOFECTIN^®. After an overnight incubation, transfected cells were fed with complete medium and allowed to recover for 48 hours. Antibiotic selection was applied by addition of G418 sulfate (250 μg/ml)

(GENETICIN^®, GIBCO) to the medium and the cells were allowed to grow for 2 weeks . Fresh medium was replaced every 3 days . The remaining viable cells were replated in complete medium without antibiotic, seeded into 6 cm dishes, and treated with vehicle, l,25-(OH)₂D₃ (1 nM) , or dexamethasone (100 nM) for

24 hours. Cell lysates were prepared and luciferase activity measured as described above.

B. Results

Establishment of permanently transfected cells that are highly responsive to vitamin D treatment was achieved by introduction of the 24-hydroxylase promoter and a portion of the 5' flanking region into an expression vector, pMAM_neo-LUC (Fig. IA) . 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.

To obtain maximal levels of 1,25-(OH)₂D₃-inducible luciferase activity, 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, designated p23, 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). 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. 2 and SEQ ID NO: 3 and 4). These 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.

Two 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 . When the radiolabeled oligonucleotides were hybridized with the recombinants, 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)₂D₃ 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. Treatment of the stable transfectants with 1, 25-(OH)₂D₃ over a range of 1 pM to 100 nM stimulated luciferase activity between 2- and 20-fold, depending on the concentration of hormone administered (Fig. 4). Dexamethasone treatment had insignificant effects on expression levels, which agrees with the observation in transiently transfected cells.

C. Creation of a Luciferase Standard Curve Permanently transformed (P-23) 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 10³ cells in 200 μL 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% C0₂, 95% humidity.

The l,25(OH)₂D₃ standards in 2 μL of 100% ethanol were added to 50 μL 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% C0₂, 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 μL 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. 10 μL of the mixture was withdrawn for the assay and added to 100 μL of substrate: (20 mM Tricine; 1.0 mM Mg(C0₃)₄Mg(OH)₂; 2.7 mM MgS0_<,; 0.1 mM EDTA; 1.0 mM DTT; 270 μM Acetyl CoA [Li Salt]; 470 μM D- luciferin; 526 μM ATP).

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.

TABLE 2

Concentration Relative Light Units per well RLU's

Lysis Buffer 2 μL 199

Dexamethasone 2 μM 357 l,25(OH D,

Standards

10.0 pg/we 11 2873

5.0 2713

2.5 1838

1.0 1386

0.5 1134

0.25 986

0.1 701

EtOH 387

Example 2-Characterization of the 1 , 25- ( OH -D 24-hydroxylase promoter .

A. Materials and Methods Synthetic Oligonucleotides.

Several synthetic 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

ORE,: -262 to -238: 5" CTAGAGAGCGCACCCGCTGAAGCCTGGGCT

TCTCGCGTGGGCGACTTGGGACCCGAGATC 5' (SEQ ID N0:5) DRE-: -154 to -134: 5' CTAGACGGCGCCCTCACTCACCTCGCT

TGCCGCGGGAGTGAGTGGAGCGAGATC 5' (SEQ ID NO:6) -154 to -125 5' CTAGACGGCGCCCTCACTCACCrCGCTGACTCCAT

TGCCGCGGGAGTGAGTGGAGCGACTGAGGTAGATC 5' (SEQ ID NO:7) ORE, no 3' half-site: 5' CTAGAGAGCGCACCCGCCTGGGCT

TCTCGCG GGGCGGACCCGAGATC 5' (SEQ ID NO:8) DRE, no 5' half-site: 5' CTAGAGAGCTCTGAACCCTGGGCT

TCTCGAGACTTGGGACCCGAGATC 5' (SEQ ID NO:9) -260 to -136: 5' - > 3' Xbal

5' CAGGCGTTCTAGAGCGCACCCGCTGAACCC (SEQ ID NO:10)

3' - > 5' Xbal

5' CAGGCGTTCTAGAGAGGTGAGTGAGGGCGC (SEQ ID NO:11 )

DRE, and DRE, separated by 93 5' - > 3' DRE, bp of nonspecific DNA: 5- GAGCGCACCCGCTGAACCTCTAGAGGTCAGCTTTCCAAGAAG

(SEQ ID NO: 12)

3' - > 5' ORE_¬ S' GCGAGGTGAGTGAGGGCGTCTAGAAGCAAACTGTTGGCCG

TC (SEQ ID NO: 13)

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.

Recombinant Reporter Constructs and Cell Transfection.

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 μg 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)₂D₃ 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,*).

All K_d determinations were done by gel retardation assays . Probes were prepared synthetically or amplified by PCR as shown above. Labeling of each probe was done by repairing Xbal recessed ends utilizing α[³²P]dCTP

(DuPont/NEN, Boston, MA) and the Klenow fragment of DNA polymerase (Promega, Madison, WI) . The probe obtained by PCR using chicken 24-hydroxylase cDNA was endlabeled utilizing γ[³²P]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/μl or 1 ng/μl. Binding of the VDR to the labeled DNA probes was performed as described previously (Darwish and DeLuca, Proc. Natl. Acad. Sci . USA 89:603-607, 1992). Briefly, 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)₂D₃. When 1, 25-(OH)₂D₃ 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)₂D₃. 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.

B. Results

Fig . 5 shows the structure of each pBLCAT2 construct used and the respective reporter gene activity induced by 1 , 25-(OH)₂D₃. Referring to Fig. 5, 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_:

(a...b) is located between -262 and -238 and DRE₂ (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 half-site at

-262 is responsive to l,25-(OH)₂D₃ by itself but to a much lesser degree than the entire DRE system.

The DRE at -154 is not responsive to l,25-(OH)₂D₃ by itself but requires the presence of a third half-site located at the 3 '-end. All three sites are required for

1, 25-(OH)₂D₃-dependent activity as shown in Fig. 6. Fig. 6 diagrams CAT activities resulting from different combinations of half-sites of DRE₂ (-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 μg 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)₂D₃ is unchanged.

Our results suggest that DREj and DRE₂ (containing a total of 5 half-sites) account for most if not all of the transactivation potential of the intact promoter of the rat 24-hydroxylase. The somewhat higher +D/-D ratio seen with the intact 24-hydroxylase promoter and a promoterless CAT reporter is likely due to the lower baseline activity found in the cells without 1,25-(OH)₂D₃. K_ds were determined for each DRE separately, and the DREs together with their natural flanking sequences (separated by 93 bp) (see Table 4, below).

Referring to Table 4, 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 .

TABLE 4

FRAGMENT -D +D d Kd

-262 to -238 Kdl 1.3±0.6 nM Kdl: 5.3±1.4nM Kd2 12.3±3.2 nM Kd2:46 nM

-154 to -134 Kdl 5.2±2.1 nM Kdl: 5.7±0.1 nM Kd2 30±7 nM Kd2: 33+19 nM

-154 to -125 Kdl 4.4±1.4 nM Kd2 30±7 nM not determined

-260 to -136 Kdl: 13.7 pM

93 bp Kd2: 0.2+0.04 nM not determined

-260 to -238 and -154 to -136 separated by 9 bp of nonspecific DNA 9₃ bp no nspecific Kd: 0.6±0.01 nM not determined

-262 to -238 no 3' half-site

Kd: 167 nM not determined

-262 to -238 no 5' half-site

Kd: 143 nM

Multiple determinations were done and a representative gel shift for each fragment was determined. Increasing amounts of the indicated DNA probes were incubated with 24 f oles of VDR. 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 DRE₂ (-154 to -134), and Figs. 7C and F represent nucleotides -260 to -136. Average K_ds (-1/slope) for several determinations are shown in Table 4. Table 4 presents the K_dS 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_ds 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 DRE₂ assayed separately, and Fig. 8B diagrams DREj and DRE₂ separated by their natural promoter sequence.

No cooperative binding of the VDR to the two half-sites of the DREs was detected (Fig. 8A) , which could have explained the biphasic binding. There also seemed to be no cooperativity of VDR binding between both DREs (Fig. 8B).

The tightest binding was observed with the intact DRE system. Substitution of the 93 bp fragment separating the two DREs with an irrelevant chicken DNA had little effect. The DRE at -268 to -238 had approximately one-hundredth the binding affinity of the two DRE systems, while the DRE at -154 to - 125 had one-five hundredth the affinity. Halfsites were bound poorly or about 10,000 times lower than the DRE complex, and were not responsible for the biphasic binding pattern, since they seemed to have equal binding affinities for the VDR.

The K_ds in the presence of ligand were also determined, and these values are also shown in Table 4. Binding proved to be 2-3-fold weaker in the presence of 1, 25-(OH)₂D₃, regardless of the salt concentration at which they were determined. However, the salt concentration at which maximal binding was achieved was higher (75 mM KC1 vs. 150 mM KC1). The fact that in the absence of ligand, less salt is needed to destabilize the DNA/receptor complex would suggest that this complex is less stable than the complex with 1,25- (OH)₂D₃, yet the dissociation constant is 2-3 times lower which indicates greater affinity. We have no explanation for this interesting anomaly.

We then compared K_ds to their respective 1,25-(OH)₂D₃- dependent transcriptional activities (Fig. 5 and Table 2). There was no such correlation between transcriptional activity and K_d for any of the DNA fragments . Therefore, we have demonstrated that the promoter region of the rat 1 , 25-(OH)₂D-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)₂D₃ 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.

The most significant fact, however, is that there are two distinct D-responsive elements found in the promoter region of this gene and both are required for maximal transactivation activity. These two DREs are separated by a 93 bp fragment which can be deleted without significantly reducing the transactivation potential of the system.

We note that the presence of two hormone responsive elements in the same promoter is unique for 1,25-(OH)₂D₃. The vitellogenin gene has two EREs (J. Burch, e_t al. , Mol.

Cell . Biol . 8:1123-1131, 1988) while the mouse mammary tumor virus (MMTV) long terminal repeat contains multiple copies of the GRE (C. Scheidereit, et al., Nature 304:749-752, 1983).

In dissecting the trans-activation potential of this system, our results demonstrate that the DRE more distal to the transcriptional start site is capable of responding in the reporter gene system to 1,25-(OH)₂D₃. The DRE proximal to the transcriptional start site did not respond to 1,25- (OH)₂D₃ until a third half-site was included. Each of the DREs alone could not approach the activity of the entire system. Finally, it is apparent from our results that the total 5 half-sites can account for virtually all of the transactivation potential of the VDR system found in the intact 24-hydroxylase promoter. Since the intact promoter was placed in a promoterless reporter gene system, 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. Furthermore, the 24-hydroxylase promoter in the promoterless CAT reporter system shows low basal activity which accounts for the higher ratio of +D/-D response.

Because of these findings, we attempted to learn whether the transactivation of each of these elements is related to their ability to bind to the receptor and the accessory protein. The K_ds determined for each component of the VDR system did not correlate with its activity in the reporter gene system. However, the tightest binding occurred with the intact double responsive element system, and it was this system that gave maximum trans-activation. In general, when examining the many oligonucleotides studied, no correlation between binding and transactivation was detected.

Example 3-Characterization of a reporter gene assay for estrogen

We envision at least two different approaches to creating an estrogen-responsive system. 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.

I A. Creation of a gene construct

Construct No. 1. Synthetic oligonucleotides were made containing sequences from the chicken vitellogenin promoter (nucleotides

-356 to -331 as numbered in Burch, e_t ajL. Nucl . Acid Res .

12:1117-1135, 1984) with Xbal overhangs. The oligonucleotides were as follows: 5' CTAGAGGTCAACATAACCTGGGCAAAACCA

TCCAGTTGTATTGGACCCGTTTTGGTGATC 3' (SEQ ID NO: 14)

We will insert two of the above estrogen response elements (ERE) in tandem in front of the thymidine kinase

(TK) promoter in pBLCAT2. We will then excise the two tandem ERE along with the TK promoter and insert the segment into the Nhel site of the promoterless p ^ -^-LUC vector described above (Clontech) .

Construct No. 2:

We will PCR amplify two promoter fragments which both contain two EREs from chicken genomic DNA . Both fragments have been shown to have highly estrogen-inducible reporter gene activity when placed in front of the TK promoter. Despite the TK promoter, the basal activity in the absence of estrogen is very low. The sequences we will amplify correspond to nucleotides -630 to -331 of the chicken vitellogenin promoter and nucleotides -1133 to -331 of the chicken vitellogenin promoter (as numbered in Burch, e_t al. Nucl . Acids Res . 12:1117-1135, 1984). Both fragments will be independently subcloned in front of the TK promoter in pBLCAT2. Then we will excise the amplified fragment with the TK promoter and subclone it into the Nhel site of the promoterless pMA -iβ_o-LUC vector.

B. Measurement of Estrogen

As described above, we will create a permanently transfected cell line with the constructs described above from cells that express the estrogen receptor. These cell lines will be used to measure levels of estrogen in biological samples as described above for vitamin D. We envision that one will be able to detect estrogen with the same level of sensitivity as was shown in the 1,25- dihydroxyvitamin D experiments .

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(i) APPLICANT: DeLuca, Hector F. Arbour, Nancy C. Prahl, Jean M. Ross, Troy K. Zierold, Claudia (n.m.i.)

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(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1825 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GGGGTCTTTC ACATGTTTTT CACGTTGAGC TGAGGTGAAA TTTGCCCCCC CCCAACCCCA 60

CGCATATACA ACAGAGAGCT GCTTTGTCTG GCTTCAGTGG GAGAGGATGT GCCTGATCCT 120

ACAGAGACTT AATACACCAG GGTGAGGGGA TATGGCGGGG GGGGGGGGCA CCCTCTCAGA 180

AGGGAAGGGG AAATGGTAGG AGGAACCTGT GAGGGCACAC GGGGAGGCGG CAGCACCGGG 240

ATGTAAACAC TAAAATAAAT TATTAATAAA TAAGAAGCAT TGGCGTGAAA TTCCTGGTTT 300

ATCACAGGCT CTCAGAAGCA TAGCATGTGG TACCCTCATG CAGTTATAAG GCCTACACTG 360

TATTACATAG AGAATAAAAT GTATGCTCCA CAGACACACA AAAGACTGTG TAAGCTTACA 420

GTGTGTGTGC CGTACACACT GCAGTGCATA TTGTAAAAGA TCTGATGGAT TTCAGCTAAA 480

TATTATTGCT TATGTGCAGC TCTTGTATTT TTTGTTTTTT GTTTTTTTTA ATTAGCGCCG 540

ACCTCTTAGG GGATATTTGA AGTGACAGCG TTGGAGCACA ATCACAAATC GGATTGCAGA 600

AGTTCTCCAG AAGCTGACAG ATGTTTACGG AAGACTTTGC TATTGCTTGG GTCACGGTAA 660

CCTCAGGTAA GGTGACTGGA AAGCAGGGGA GGAAGACTCT GCCTCCTGCC AGGGTGCTGG 720

TGGCTTTGCA TTGGAAAAGC GGGAAACAAT ATGAATCACT TTGAGTCAGC CGAGACCAAT 780

TTCCTTTCCA GAATTGGAAA GCGAGCTCTG TTCTATCCGG CCGCAAAAGC AGGAGTGCGA 840

CTGTGATAAC CCTGCCTGCT TTAGGTGGGC TTTAGGGAGA AAGTGGGGCT CTTGGGAACC 900

TGTGGGTGCC TCTGCTCTGT GCAGGGTGGC GGTGCGCAAG AAGGAAAGGC CCGAGGGACC 960

TGTGGGAAAC AGTCTAAAGG AAACTGAGCT AGTCCCTGTA GGCATTGCAC AGTCTCCGGA 1020

AGAACTAAGG CCACTAGTAT CCTTTATTGA GGACACACAC CTGTGTAGAC CTTACATGCG 1080

TTCATTTATT CGATTCCCTA ACAAGTCAAC CCGAAGCATC GAGGAATCTG GTAAGGAAAT 1140

TCTGCAAACG CATTGGGCTT TCTTGCCTAA TTTAAGACCC AAGGTAAGAA GATTATTTCC 1200

TCTCCCCCTC CCCTCTTTTT GGTATAGCCT AGTAAGTTTC AAGTCCTCTC TTCCTTCAGA 1260

AGCTCAAAAA GGCAACTTTC ATCCAAGGGA AGTCTGGCTC AGGCTGCGAA AACCTGTCTC 1320

CCAGGATATT GGAAGGCGGA CACTCTCCAC TTGCTTGGCA AGCGCGGGCG GCCTTCCGCG 1380

CTGTCCTCAG GGACCTTGCC CGCCCTGCAT GGCGATTGTG CAAGCGCACG TTTGGGCTCC 1440

TGCAAGGCCA GCTGCAGCCT GCAGGAGGAG AGCGAGGAGG CGTGCTCGAG CGCACCCGCT 1500

GAACCCTGGG CTCGACCCGC CTTTCTCAGG TTATCTCCGG GGTGGAGTCC ACCGGTGCGT 1560

CTGCCGGGCC AGCAGCGTGT CGGTCACCGA GGCCCCGGCG CCCTCACTCA CCTCGCTGAC 1620 TCCATCCTCT TCCCACACCC GCCCCCCGCG TCCCTCCCAG CCGGTCCCCC TCCGCCCTGC 1680

TCAGCGTGCT CATTGGCCAC TCCAGCATGC CCTGCTTCCA TAAATACGAG GTCCCTATGC 1740

CTGGAATCTT GGGGAGGTAC AACGAGGTAC ATCAAACCTG GCGGCATCCC TTCGACCCTC 1800

CTTGATTCCC CCGTGGCTTT AGGCC 1825

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CGCACCCGCT GAACC 15

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGGTACCGAG CTCTGTTCTA 20

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GTACCGAGCT CTGTTC 16 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CTAGAGAGCG CACCCGCTGA ACCCTGGGCT 30

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CTAGACGGCG CCCTCACTCA CCTCGCT 27

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTAGACGGCG CCCTCACTCA CCTCGCTGAC TCCAT 35

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CTAGAGAGCG CACCCGCCTG GGCT 24

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CTAGAGAGCT CTGAACCCTG GGCT 24

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CAGGCGTTCT AGAGCGCACC CGCTGAACCC 30

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CAGGCGTTCT AGAGAGGTGA GTGAGGGCGC 30 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GAGCGCACCC GCTGAACCTC TAGAGGTCAG CTTTCCAAGA AG 42

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GCGAGGTGAG TGAGGGCGTC TAGAAGCAAA CTGTTGGCCG TC 42

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CTAGAGGTCAACATAACCTGGGCAAAACCA 30

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other Nucleic Acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GGGTGTCGGA AGCCC 15

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other Nucleic Acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GGGTGAATGA GGACA 15

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other Nucleic Acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GGGTGAACGG GGGCA 15

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other Nucleic Acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GGTTCACGAG GTTCA 15 (2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other Nucleic Acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGTTCAGCGG GTGCG 15

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: 1inear

(ii) MOLECULE TYPE: Other Nucleic Acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: GGGGGATGTG AGGAG 15

Claims

ClaimsWe claim:

1. A method for the quantitative detection of a hormone, comprising the steps of

(a) exposing a sample of a substance containing a hormone to a cell line transformed with a gene construct,

.wherein the gene construct comprises a promoter comprising a hormone response element operably connected to a reporter gene and wherein said hormone response element is responsive to said hormone, and

(b) measuring the expression level of the reporter lQene and correlating the expression level with hormone concentration.

2. The method of claim 1 wherein the hormone is selected from the group of steroid, thyroid and retinoid hormones .

3. The method of claim 1 wherein the reporter gene encodes luciferase.

4. A method for the quantitative detection of vitamin 1,25-dihydroxyvitamin D₃ or a 1-hydroxyvitamin D analog, comprising the steps of

(a) exposing a sample of a substance containing at SLeast one of 1,25-dihydroxyvitamin D₃ and analogs thereof to a cell line transformed with a gene construct, wherein the gene construct comprises a promoter containing a vitamin D response element operably connected to a gene encoding a reporter gene, and 10 (b) measuring the expression level of the reporter gene and correlating the expression level with 1,25- dihydroxyvitamin D₃ concentration.

5. The method of claim 4 wherein the promoter is the 24-hydroxylase promoter.

6. A gene construct comprising a promoter operably connected to a gene encoding luciferase, wherein the promoter comprises a vitamin D response element found in the 24- hydroxylase promoter.

7. The construct of claim 6 wherein the hormone response element is responsive to molecules selected from the group consisting of l,25(OH)₂D₃ and analogs thereof.

8. The construct of claim 6 wherein the hormone response element is responsive to 1,25(OH)₂D₃.

9. The construct of claim 6 wherein the promoter contains nucleotides -1399 through +76 of the 24-hydroxylase gene.

10. The construct of claim 6 wherein the promoter contains nucleotides -262 through -125 of the 24-hydroxylase promoter.

11. The construct of claim 6 wherein the promoter contains nucleotides -262 through -238 of the 24-hydroxylase promoter.

12. The construct of claim 6 wherein the promoter contains nucleotides -154 through -125 of the 24-hydroxylase promoter.

13. The construct of claim 6 wherein the construct is part of a plasmid comprising endogenous promoters and the construct promoter and luciferase gene are in an orientation opposite that of endogenous plasmid promoters.

14. A cell line permanently transformed with the construct of claim 6.

15. A cell line permanently transformed with the construct of claim 9.

16. A cell line permanently transformed with the construct of claim 10.

17. A cell line permanently transformed with the construct of claim 11.

18. A kit for quantitatively determining the amount of a hormone in a substance, comprising: a receptacle containg a cell permanently transformed with a gene construct comprising a promoter operably connected to a reporter gene, wherein the promoter comprises a hormone response element and wherein the hormone response element is responsive to said hormone, and a receptacle containing reagent necessary to analyze expression of the reporter gene.

19. A kit for quantitatively determining the amount of l,25(OH)₂D₃ in a substance, comprising: a receptacle containg a cell permanently transformed with a gene construct comprising a promoter operably connected to a reporter gene, wherein the promoter comprises a vitamin D response element, and a receptacle containing reagent necessary to analyze expression of the reporter gene.