Cross Reference to Related Applications This application is a continuation of and claims priority from U. S. Provisional Patent Application Serial No. 60/093, 778, entitled"Use of FGF-BP Gene Promoter Sequences as Sensors of Drug Effects and Oncogenic, Angiogenic, Vascular, and Immune Function,"filed July 23,1998.

Background of the Invention 1. Field of the Invention The present invention relates to nucleic acid sequences encoding FGF-BP promoters, and to methods of using these sequences as sensors of drug effects and oncogenic, angiogenic, vascular, and immune function, as well as in the diagnosis and treatment of diseases involving the abnormal expression of FGF-BP, such as cancers expressing FGF-BP. The sequences and methods of the present invention are also useful to promote angiogenesis, to facilitate wound healing and in other applications.

2. Description of the Background FGF-BP is a secreted protein which binds to aFGF and bFGF in a non-covalent reversible manner [1]. FGF-BP mRNA has been found to be up-regulated in squamous cell carcinoma (SCC) cell lines of different origin, in SCC tumor samples from the head and neck, and in some colon cancer cell lines [1, 2]. More recently, developmental expression of the mouse FGF-BP gene was found to be prominent in the skin and

intestine during the perinatal phase and is down-regulated in adult mice [3]. It has been shown that expression of FGF-BP in a non-tumorigenic human cell line (SW-13) which expresses bFGF leads to a tumorigenic and angiogenic phenotype [2]. Expression of FGF-BP in these cells solubilizes their endogenous bFGF from its extracellular storage and allows it to reach its receptor suggesting that FGF-BP serves as an extracellular carrier molecule for bFGF [2, 4]. Expression of FGF-BP under the control of a tetracycline-responsive promoter system in SW-13 cells revealed its role during the early phase of tumor growth [5]. To assess the significance of FGF-BP endogenously expressed in tumors, human SCC (ME-180) and colon carcinoma (LS174T) cell lines were depleted of FGF-BP by targeting with specific ribozymes [6]. This study showed that the reduction of FGF-BP reduced the release of biologically active bFGF from cells in culture. In addition, the growth and angiogenesis of xenografted tumors in mice was decreased in parallel with the reduction of FGF-BP suggesting that some human tumors can utilize FGF-BP as an angiogenic switch molecule.

Summary of the Invention The fact that FGF-BP has been detected in only a few types of tumors where it seems to play a crucial role in angiogenesis led us to investigate the mechanisms responsible for turning its expression on or off. By studying the regulation of FGF-BP in SCC cell lines, it has been found that all-trans-retinoic acid (tRA), used as a chemotherapeutic agent against SCCs, down-regulates FGF-BP gene expression in vitro by both transcriptional and post-transcriptional mechanisms [7]. In vivo tRA treatment reduces FGF-BP expression in SCC xenografts and inhibits their tumor growth and angiogenesis [8]. On the other hand, FGF-BP mRNA expression in the adult mouse skin was found to be dramatically increased during the early stages of DMBA/TPA-induced mouse skin papilloma formation [3], as well as in DMBA/TPA-treated human skin grafted onto SCID mice (unpublished data). Similarly, FGF-BP expression in vitro was

up-regulated in epidermal cell lines carrying an activated ras gene, implicating the ras/PKC pathway in the regulation of FGF-BP [3].

In this context, and given the fact that FGF-BP could play a critical role in the development of human skin cancer, the effects of the tumor promoter TPA on FGF-BP gene regulation were investigated. It has been discovered that FGF-BP mRNA expression is up-regulated by TPA in the ME-180 SCC cell line and that this induction is mediated by direct transcriptional mechanisms. Analysis of the human FGF-BP promoter reveals that the TPA induction is mediated by cooperation of several inducible regulatory elements. Furthermore the induction of FGF-BP gene expression by TPA can be modified by a repressor element juxtaposed to the AP-1 site which contains sequences recognized by E-box element factors.

The present invention is directed to FGF-BP promoters and related nucleic acid sequences, and to methods of using these sequences as sensors of drug effects and oncogenic, angiogenic, vascular, and immune function and in the diagnosis and treatment of diseases, such as those involving the abnormal expression of FGF-BP, including cancers expressing FGF-BP.

One embodiment of the invention is directed to a nucleic acid comprising an FGF-BP promoter sequence, the sequence comprising an effective portion (preferably at least 10 nucleotides in length) of the sequence specified in SEQ ID NO : 1, and a target gene, such as a detectable marker. The promoter sequence is preferably transcriptionally linked to the target gene.

Another embodiment is directed to a method for detecting an agent that inhibits FGF-BP transcription comprising the steps of delivering to a cell a nucleic acid comprising an FGF-BP promoter sequence, the sequence comprising an effective portion of the sequence specified in SEQ ID NO : 1 and a gene encoding a detectable marker, such that the nucleic acid molecule is transcribed, adding the agent to the cell,

and detecting whether or not the agent inhibits transcription of the detectable marker by detecting the absence or presence of the marker.

Another embodiment is directed to a method for detecting an agent that induces FGF-BP transcription comprising the steps of delivering to a cell a nucleic acid comprising an FGF-BP promoter sequence, the sequence comprising an effective portion of the sequence specified in SEQ ID NO : 1 and a gene encoding a detectable marker, such that the nucleic acid molecule is transcribed, adding the agent to the cell and detecting whether or not the agent induces transcription of the detectable marker by determining the absence or presence of the marker.

Another embodiment is directed to a compound that inhibits transcription of an FGF-BP gene comprising an agent that inhibits FGF-BP promoter function.

Another embodiment is directed to a compound that induces transcription of an FGF-BP gene comprising an agent that induces FGF-BP promoter function.

Another embodiment is directed to a therapeutic compound for use in the treatment of diseases characterized by abnormal FGF-BP expression comprising an agent that alters transcription or expression of FGF-BP. Depending on the application, the agent may induce or inhibit transcription or expression of FGF-BP.

Other embodiments of the invention are directed to an isolated nucleic acid comprising SEQ ID NO : 1, and to isolated and purified nucleic acid sequences comprising all or an effective fragment of SEQ ID NO : 1.

Another embodiment is directed to a diagnostic kit for detecting FGF-BP transcription comprising all or a portion of an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1. The sequence may be linked to a detectable label that is radioactive or non-radioactive.

Another embodiment is directed to a nucleic acid which hybridizes preferentially to all or a portion of the nucleotides of the nucleic acid sequence of SEQ ID NO : 1.

Another embodiment of the invention is directed to a nucleic acid probe that hybridizes to a region of the FGF-BP gene.

Another embodiment is directed to a method for treating diseases characterized by abnormal FGF-BP expression by administering a composition comprising a pharmaceutically effective amount of an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1. The composition may include other therapeutic agents or factors, such as tumor suppressor gene.

Another embodiment is directed to a composition comprising an expression vector including a constitutively active promoter and an antisense FGF-BP promoter sequence wherein the antisense FGF-BP promoter sequence is transcribed into antisense RNA.

Another embodiment of the invention is directed to a vector comprising a constitutively active promoter functionally linked to an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1.

Another embodiment is directed to a composition for the treatment or prevention of diseases characterized by abnormal angiogenesis comprising a recombinant vector containing an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1 functionally linked to a desired gene, such as a tumor suppressor gene.

Other objects and advantages of the invention are set forth in part in the description which follows, and in part, will be obvious from this description, or may be learned from the practice of the invention.

Description of the Drawings Figure 1A. Time dependence of FGF-BP mRNA induction after exposure to 10-7M TPA.

Figure 1B. Dose-dependent TPA Effects on FGF-BP mRNA.

Figure 1C. The effect of Calphostin C on the TPA-induction of FGF-BP mRNA. ME- 180 cells Figure 2A. Effect of Actinomycin D and Cycloheximide on FGF-BP mRNA induction by TPA.

Figure 2B. Analysis of turnover of FGF-BP mRNA in TPA-treated cells.

Figure 3. Transcription run-on analysis of the effects of TPA on FGF-BP gene expression.

Figure 4. Mapping of the FGF-BP transcription start site.

Figure 5A. 1. 8 kb human FGF-BP promoter region, GenBank Accession #AF062639.

(1891 base pair- human).

Figure 5B. Structure and homology of the human FGF-BP promoter.

Figure 6. Effect of progressive 5'deletions on basal activity and TPA-induction of FGF-BP promoter.

Figure 7. Effect of internal deletions on basal activity and TPA-induced activity of the FGF-BP promoter.

Description of the Invention As embodied and broadly described herein, the present invention is directed to nucleic acid sequences, and more particularly, to FGF-BP promoters, and to methods of using these sequences as sensors of drug effects and oncogenic, angiogenic, vascular, and immune function. The sequences are also useful in the diagnosis and treatment of diseases, such as those involving the abnormal expression of FGF-BP, including cancers expressing FGF-BP.

The following abbreviations have the following meanings as used herein : TPA, 12-0-tetradecanoyl-phorbol-13-acetate ; SCC, squamous cell carcinoma ; FGF-BP, fibroblast growth factor binding protein ; FGF, fibroblast growth factor ; tRA, all-trans- retinoic acid ; DMBA, 7,12-dimethylbenz[a]anthracene; PKC, protein kinase C ; C/EBP, CAATT enhancer binding protein.

As noted, given the fact that it was possible for FGF-BP to play a critical role in the development of certain cancers, such as human skin cancer, the effects of the tumor promoter TPA on FGF-BP gene regulation were investigated. As discussed below, it has been discovered that FGF-BP mRNA expression is up-regulated by TPA in the ME- 180 SCC cell line and that this induction is mediated by direct transcriptional mechanisms. Analysis of the human FGF-BP promoter reveals that the TPA induction is mediated by cooperation of several inducible regulatory elements. Furthermore the induction of gene expression by TPA can be modified by a repressor element juxtaposed to the AP-1 site which contains sequences recognized by E-box element factors.

TPA increases FGF-BP mRNA in SCCs. An up-regulation of FGF-BP mRNA following TPA treatment of mouse skin during the development of skin tumors [3] and also in human skin xenografts has been previously detected (unpublished observation).

This data suggests that the control of this angiogenic switch factor may play an important role in skin carcinogenesis. To examine this further the effect of the tumor promoter TPA on FGF-BP gene expression in ME-180 cells which express high levels of the FGF-BP transcript was studied [7].

Northern blot analyses were performed with 30 pg of total RNA/lane. Bands corresponding to FGF-BP mRNA (1. 2 kb) and the control gene GAPDH mRNA (1.3 kb) were probed. Figures 1A, 1B and 1C show the quantitation of the Northern blot data. Signal intensities were quantified by phosphor-imaging and normalized to the control gene, GAPDH. The meanSD of 2 separate experiments is given. Cells were treated with 10-7 M TPA from 1 to 24 hours which resulted in an increase in the steady- state levels of FGF-BP mRNA detectable 1 hour after treatment. Phosphor-Imager analysis showed that the induction was maximal after 6 hours by 452 +/- 44 %. (Figure 1A,). GAPDH mRNA remained unaffected by TPA treatment, as judged relative to the total amount of RNA loaded and was used to standardize FGF-BP mRNA. The dose-

dependence of TPA induction of FGF-BP mRNA in ME-180 is shown in Figure 1B.

The half-maximal effective concentration of TPA is estimated to be 1 nM. The inductive effect of TPA on FGF-BP mRNA was also observed in two other SCC cell lines, FaDu and A431 (data not shown) demonstrating that TPA induction of FGF-BP mRNA is generally preserved in SCC cell lines.

To establish whether TPA induction of FGF-BP mRNA was mediated through a PKC-dependent pathway, ME-180 cells were pretreated or not pretreated for 2 hours with 100 nM of the highly specific PKC inhibitor, Calphostin C [13], and then treated with or without 10-7M TPA for 4 hours. As can be seen in Figure 1C, pretreatment of the cells with Calphostin C prior to TPA treatment totally blocked the TPA effect demonstrating that the induction of FGF-BP transcript by TPA is mediated via a PKC- dependent mechanism. In addition, it is known that TPA causes an immediate up- regulation of PKC followed by long term down regulation of PKC activity. In contrast, although long-term Calphostin C appears to be able to down-regulate protein kinase C it does not cause early induction of PKC activation but rather blocks the inductive effects of TPA on PKC [14]. Thus the fact that Calphostin C causes no induction of FGF-BP mRNA and blocks the TPA effect argues that induction of FGF-BP mRNA is through an up-regulation of PKC activity rather than a consequence of long-term down- regulation.

Mechanism of TPA induction of FGF-BP mRNA. We have previously shown that FGF-BP gene expression can be regulated through both transcriptional and post- transcriptional mechanisms [7]. Therefore we next attempted to determine whether the TPA induction of FGF-BP mRNA was at the transcriptional or post-transcriptional level. We first assessed whether TPA treatment affected the stability of the FGF-BP mRNA. Experiments were performed to determine whether addition of inhibitors of transcription (actinomycin D) or translation (cycloheximide) could inhibit the TPA induction of FGF-BP mRNA. ME-180 cells were treated with 5 tg/ml actinomycin D

(ActD) or 10 ug/ml cycloheximide (CHX) in the absence or presence of 10-7 M TPA, and FGF-BP mRNA levels were determined 6 hours after treatment. (Figure 2A).

Northern blot analyses were performed as described in Example 1. Figures 2A and 2B represent quantification of the Northern blots. Signal intensities were quantified by phosphor-imaging and normalized to GAPDH. Results represent meanSD of two independent experiments.

As shown in Figures 2A and B, simultaneous addition of TPA and actinomycin D completely blocked the TPA induction whereas simultaneous addition of TPA and cycloheximide had no effect. These data suggest that TPA directly increased the rate of FGF-BP gene transcription independently of de novo protein synthesis and did not affect the stability of the FGF-BP transcript. To further verify that the stability of the FGF-BP transcript was not modified by TPA treatment, ME-180 cells were pretreated with TPA and then actinomycin D was added to inhibit transcription. Specifically, ME- 180 cells were treated with vehicle alone or 10-7M TPA for 2 hours, and 5 ng/ml actinomycin D was then added to control-treated and TPA-treated cells for 0-16 hours (Figure 2B). Total RNA was isolated and hybridized sequentially with FGF-BP and GAPDH probes as described in Example 1. As shown in Figure 2B, pretreatment of cells with TPA did not increase the half-life of the FGF-BP mRNA indicating that the stability of the FGF-BP transcript is not affected by TPA.

To directly prove that TPA increases the rate of transcription of the FGF-BP gene, we then performed nuclear transcription run-on assays. ME-180 cells were treated with or without 10-7 M TPA for various periods of time. P32-Labeled nascent transcripts were prepared from isolated ME 180 cell nuclei and hybridized to a nylon membrane bearing immobilized target DNA sequences. As shown in Figure 3, TPA increased FGF-BP transcript levels maximally after 1 hour of treatment. Quantitation and normalization to B-actin showed that FGF-BP transcription was up-regulated by

647 +/- 1,448 +/- 16, and 197 +/- 31% after 1,4, and 24 hours of treatment, respectively (Figure 3). B-actin plasmid DNA was used as a control since transcription of this gene remained constant. These findings are consistent with the above results studying steady-state mRNA and the effects of actinomycin D and cycloheximide treatment. Clearly the induction of FGF-BP mRNA by TPA in ME-180 cells is directly due to a rapid up-regulation of transcription.

Isolation and Characterization of the Human FGF-BP Promoter. In order to better understand the transcriptional regulation of the human FGF-BP gene, 1. 8kb of genomic sequence upstream to the known 5'UTR sequence of human FGF-BP cDNA was isolated from a human genomic library and sequenced. The complete 1. 8kb sequence has been deposited with GenBank under accession #AF062639 and is given as SEQ ID NO : I in Figure 5A. The structure of the human FGF-BP promoter from -1829 to +62 is shown schematically as a straight line in Figure 5B. The human and mouse FGF-BP promoters were found to share a region of homology. The region of homology between the human and mouse FGF-BP promoter sequences is shown in Figure 5B, in which nucleotides -250 to +62 (SEQ ID N0 : 2) of the human FGF-BP promoter are aligned with nucleotides -265 to +51 (SEQ ID N0 : 3) of the mouse FGF-BP promoter.

Transcription start sites are indicated by an asterix. Vertical lines indicate homologous nucleotides between the mouse and human and dotted lines represent gaps in the homology. Consensus transcription factor binding sites are boxed. Numbers on the top strand correspond to the numbering of the human FGF-BP promoter and show the location of the distal end points used to create the luciferase promoter constructs used to obtain the data shown in Figure 6.

The transcription start site of the human gene was determined by primer extension analysis using nested primers derived from known cDNA. Figure 4 schematically indicates the location of primers 1 and 2 derived from the coding region (FGF-BP primer 1) (lanes 3 and 4 on the gel, data not shown) and 5'UTR (FGF-BP

primer 2) (lanes 5 and 6 of the gel, data not shown) of the human FGF-BP cDNA. The extension reaction was carried out in the absence (lanes 3 and 5 of the gel, data not shown) or presence (lanes 4 and 6 of the gel, data not shown) of mRNA isolated from ME-180 cells. A control primer was used in the absence (lane 1 of the gel, data not shown) or presence (lane 2 of the gel, data not shown) of 1. 2kb kanamycin control RNA. The transcription start site derived from these results is depicted with an asterix in Figure 5B. Alignment between the human and mouse FGF-BP promoter which we cloned previously [3] revealed a region of high homology with 70% nucleotide identity within the first 200 nucleotides upstream from the transcription start (Figure 5B).

Nucleotide homology dropped significantly in more upstream sequences, suggesting that the proximal conserved 200 nucleotides of the promoter could be important for transcriptional regulation of FGF-BP in both species.

Sequence analysis of the promoter demonstrated the presence of numerous consensus transcription factor binding sites which were conserved between mouse and human FGF-BP promoters and which may have functional importance in FGF-BP regulation. As shown in Figure 5B, a consensus TATA box is located at about -25 upstream from the transcription start for both promoters. Between -48 and -40 of the human FGF-BP promoter we found a highly conserved consensus binding site for C/EBPB, a member of the CCAAT/enhancer binding protein (C/EBP) family of leucine zipper transcription factors which plays a central role in the acute-phase response and in a number of cell differentiation pathways [15-17]. An AP-1 consensus binding site (-65 to -59) lies juxtaposed to a sequence with homology to an Ets factor binding motif (-76 to -68), suggesting potential functional similarity to the juxtaposed Ets/AP-1 site found in the Polyoma virus enhancer and in the collagenase promoter [18, 19]. In addition, a consensus Spl factor binding site (-90 to -80), an additional Ets factor binding motif (- 107 to -100), and a potential NF-DB binding site (-185 to -176) are located in the

conserved region of the promoter and may play a role in transcriptional regulation of FGF-BP as well.

Functional analysis of the human FGF-BP promoter. To identify the functional promoter elements involved in FGF-BP gene regulation by TPA, progressive 5'deletion mutants were constructed based on the location of consensus factor binding sites on the promoter.

Each promoter construct was transiently transfected into ME-180 cells and luciferase activity is expressed as fold induction of TPA-treated over untreated for each construct.

(Figure 6). The histogram on the left in Figure 6 indicates the impact of the promoter deletion on the basal activity of each construct. The basal activity of the -118/+62 construct was set at 100%. The control vector shown is the thymidine kinase minimal promoter in PXP1. The histogram on the right in Figure 6 shows the transcriptional activity in the presence of TPA 10-7M for each FGF-BP promoter deletion construct (center). The mean basal activity of the -118/+62 construct was 15, 000 light units per ug protein and the mean TPA-induced level was 100, 000 light units/ug protein. Values represent the meanSE from at least three separate experiments, each done in triplicate wells. Asterix indicates significant difference (p<0. 05) from the -118/+62 promoter construct. The empty PXP1 vector had no detectable luciferase activity either in the absence or presence of TPA (data not shown). However, we did observe a background, approximately 2-fold, TPA induction of the PXPI vector when several unrelated minimal promoters (i. e. thymidine kinase minimal promoter, the CMV minimal promoter and the POMC minimal promoter) were treated with TPA after transfection into ME-180 cells (Figure 6 ;'Control Vector'). Background induction by TPA of a variety of vectors has been described previously and is presumably mediated through cryptic sites in the PXP 1 plasmid [20]. The TPA induction due to the inserted FGF-BP promoter was considered to be that observed above the background control vector induction. The FGF-BP promoter from -1060 to +62 was induced about 4 fold above

control vector in the presence of TPA and showed the same TPA inducibility as the full length 1. 8 kb promoter construct (data not shown). Deletion from -1060 to -118, which removed 950 bp of promoter sequence including the potential NF- 0 B site, had no effect on TPA induction and was also induced 5 fold above background (Figure 6). Similarly, deletion from -118 to -93, which removed one of the potential Ets binding sites, retained full TPA induction. Removal of the consensus Spl binding site from -93 to -77 had no effect on TPA induction of the FGF-BP promoter. However, the 5'deletion to -77 caused an 80% decrease in basal activity of the promoter (Figure 6, left panel) suggesting that the Spl consensus site is a predominant mediator of basal promoter activity of FGF-BP but is not required for TPA induction.

TPA induction was significantly reduced upon deletion of the potential Ets factor binding site from -77 to -67, and is reduced even further upon deletion of the AP- 1 site from -67 to -56 (Figure 6), indicating that each of these sites contribute to some degree in TPA induction. The basal activity of these constructs was similar at about 10- 20% of the -118 construct. Finally, deletion from -56 to -31, which removes sequences containing homology to a C/EBPB binding site, abolished any remaining TPA induction to the background control vector level. The basal activity of this vector was 5% that of the -118/+62 vector.

Contribution of Ets, AP-1 and C/EBPJJ sites to TPA induction. In order to better understand the contribution of each individual consensus binding site to TPA induction, internal promoter deletions were introduced and tested for TPA inducibility within the context of the promoter from -118 to +62 (Figure 7). Each promoter construct was transiently transfected into ME-180 cells and luciferase activity is expressed as fold induction of TPA-treated over untreated for each construct. Values represent the meanSE from at least three separate experiments, each done in triplicate wells. Asterix indicates significant difference (p<0. 05) from the -118/+62 promoter construct. The histogram on the left in Figure 7 shows the impact of each mutation on

basal activity of the construct (shown in the center of Figure 7). The control vector shown is the thymidine kinase minimal promoter in PXP1. The histogram on the right in Deletion of the Ets site alone (-76 to -67) or deletion of the AP-1 site alone (-65 to - 58) reduced TPA induction slightly to the intact promoter. Deletion of both Ets and AP-1 (-76 to -58), however, resulted in a significant decrease in both basal activity and in TPA induction, indicating that both sites act in cooperation for full promoter activity.

However, loss of the juxtaposed Ets/AP-1 site does not completely abolish TPA induction, suggesting that additional sites are also involved.

The contribution of the C/EBPB binding motif to TPA induction of the FGF-BP promoter was determined by an internal deletion from -47 to -3 (Figure 7'C/EBPB').

Consistent with the 5'deletion construct which contained only the C/EBPB site and retained some TPA inducibility (Figure 6 ;'-56/+62'), an internal deletion of this site showed a significant decrease in TPA induction. Activation of C/EBPB has been shown to occur through ras-dependent phosphorylation, and is involved in phorbol ester induction of genes such as MDR1 [21-23]. Similarly, C/EBPB seems to play a role in TPA induction of the FGF-BP promoter since deletion of this site reduces the overall induction by TPA.

TPA regulation of the FGF-BP promoter involves a repressor element juxtaposed to the AP-1 site. Between the AP-1 site and the C/EBPB site lies a region of low homology between the human and mouse FGF-BP promoter sequences. Because this region was not suspected to have any effect on TPA induction, an internal deletion removing this region (-57 to -47) was tested as a control. Surprisingly, in the -57/-47 construct, TPA induction of the FGF-BP promoter increased from approximately 5- fold to 11-fold, suggesting the presence of a possible repressor which may interact with this site. The lack of sequence conservation between the human and mouse in this region may reflect a difference in the regulation of FGF-BP between the two species. The -57 to -47 deletion disrupts an AACGTG (-60 to -55) which is juxtaposed to the 3'end of

the AP-1 site and which shows some similarity to the CACGTG E-box element recognized by a number of helix-loop-helix factors [24]. To test this imperfect E-box for repressor activity, a C to T point mutation at position -58 was introduced into the - 118/+62 FGF-BP promoter construct (Figure 7). The m-58 construct showed a dramatic increase in TPA induction up to 16-fold above background. Moreover, when the -58 point mutation is introduced into the C/EBPB construct (Figure 7, C/EBPB/m- 58'), this promoter mutant also showed increased fold induction by TPA, suggesting that repression mediated by this site is not dependent on the C/EBPB site. This data shows that the point mutation at position -58, as well as the internal deletion from -57 to -47, disrupts repression of the FGF-BP promoter which normally limits the response to TPA.

Transcription factor binding to FGF-BP promoter elements. In order to ascertain that TPA-induction of FGF-BP was due to direct activation by transcription factors, we performed gel retardation analysis to show transcription factor binding to FGF-BP promoter elements. Binding reactions were incubated in the presence of 20- fold (lane 4, of gel, not shown) or 50-fold (lane 5 of gel, not shown) molar excess of the unlabeled -80/-63 oligonucleotide. Using 32P-labeled FGF-BP promoter sequence from - 80 to -63 containing the putative Ets-site either alone (lane 1 of gel, not shown) or in the presence of untreated (lane 2 of gel, not shown) or TPA-treated (lanes 3-5 of gel, not shown) ME-180 nuclear extractsthe binding of three specific protein complexes in the presence of ME-180 nuclear extracts was detected. Protein binding to all three complexes was increased in the presence of TPA and was specifically competed away in the presence of excess unlabeled -80/-63 oligonucleotides. Further competition analysis showed that the factors binding to the -80/-63 element were only weakly competed by consensus Ets elements from the Collagenase promoter [25] and Polyoma virus enhancer [26], requiring over 100-fold excess in order to compete for binding (data not shown). It has previously been described that specific residues flanking the GGA trinucleotide motif of the Ets site are required for high-affinity sequence-specific

binding of individual Ets family members [27-29]. Therefore, our data suggests that the - 80/-63 element on the FGF-BP promoter could bind an Ets family member other than the Ets-1 or Ets-2 proto-oncogenes [26].

To determine transcription factor binding to the C/EBPB site, gel shift analysis was containing the putative C/EBPB binding site either alone or in the presence of untreated or TPA-treated ME-180 nuclear extracts. Competition for binding was carried out with 20-and 50-fold molar excess of unlabeled -55/-30 oligonucleotide. In the presence of ME-180 extracts, the -55/-30 element bound one predominant complex, which demonstrated increased binding in the presence of TPA. The majority of the complex was competed away in the presence of excess unlabeled -55/-30 oligonucleotide, indicating that binding was specific. Competition by the C/EBPB site from the p2 1WAFI/CIPI gene promoter [30] for the specific complex was effective only at high molar excess (data not shown) indicating that the FGF-BP -55/-30 element may bind a different C/EBPB-related factor.

To investigate further the transcriptional activation of the FGF-BP promoter by AP-1 and the involvement of the variant E-box repressor element, gel shift experiments were carried with labeled promoter sequence from -70 to -51 containing the AP-1 and repressor sites either alone or in the presence of untreated or TPA-treated ME-180 nuclear extracts. Competition in the presence of 10-fold, 20-fold or 50-fold molar excess of unlabeled oligonucleotides as indicated. Specific complexes are indicated by an arrow on the left of each panel and non-specific complexes are labeled"NS". In the presence of ME-180 nuclear extracts, the -70/-51 element bound an upper complex and a lower doublet. The binding of all three complexes was highly induced by TPA, and was effectively competed by molar excess of the unlabeled -70/-51 oligonucleotide. To better understand the specific composition of these complexes, point mutations were introduced in either the AP-1 site (mut AP-1) or the repressor site (mut -58) and tested for their abilities to compete for binding. Competition with the mutant AP-1 site

resulted in a decrease of only the bottom doublet and no competition for the upper band (lanes 6-8, data not shown), suggesting that the upper band corresponds to factors bound specifically to the AP-1 site. Conversely, when competition was carried out with the repressor mutant (mut -58), binding of the doublet on the probe remains intact whereas binding to the AP-1 site is reduced (lanes 9-11), indicating that the lower two bands represent distinct protein binding to the repressor element. Furthermore, when competition was carried out with an AP-1 consensus, which contains an AP-1 site flanked by sequences which are not homologous to the FGF-BP promoter, competition for only the upper AP-1 complex was observed. These results show that the AP-1 site and the repressor site of the FGF-BP promoter bind distinct and specific transcription factor complexes that are induced in the presence of TPA. Taken together, our data shows that sequences between -77 and -33 of the FGF-BP promoter form a novel TPA regulatory cassette consisting of interacting positive and negative control elements.

We have demonstrated herein that TPA induction of FGF-BP mRNA levels is primarily through stimulation of gene transcription. This is in contrast to the retinoid repression of FGF-BP gene expression which we have previously shown is. mediated through post-transcriptional and transcriptional mechanisms [7]. In fact, at least at early time points after retinoid administration, the post- transcriptional mechanism which is dependent on new protein synthesis predominates since the half life of the FGF-BP mRNA is greater than 16 hrs [7]. Our studies show that the TPA induction of FGF-BP mRNA is rapid, requiring no new protein synthesis and involves direct activation by transcription factors whose site of action is clustered in the first 118 bp upstream of the transcription start site. Within this region the majority of the TPA stimulation of the FGF-BP promoter can be explained by the additive effects of two sites positioned between -76 to -58 and from -47 to -33.

The -76 to -58 site harbors a perfect consensus to the AP-1 transcription factor binding site NTGAGTC/A [31]. The AP-1 transcription factor complex comprises the

c-fos and c-jun proto-oncogenes which are known to be activated as a result of TPA stimulation of PKC dependent pathways [32]. However, deletion of the AP-1 site alone in the FGF-BP promoter caused only a slight reduction in TPA effects on the FGF-BP promoter. This result is consistent with the emerging picture that AP-1 acts synergistically with other transcription factors, such as the Ets family of transcription factors, to mediate gene expression in response to TPA and other stimuli [28, 29]. In the FGF-BP promoter deletion of sequences 5'to the AP-1 consensus significantly decreases the TPA stimulation in comparison with deletion of the AP-1 site alone.

These 5'sequences contain the core GGA found in the center of the Ets family DNA consensus recognition site [29]. Considering the body of evidence that suggests that Ets/AP-1 co-operate for full transcriptional activation it seems likely that this may be the function of the -76/-58 element. For instance similar co-operation between Ets and AP-1 occurs through a juxtaposed Ets/AP-1 binding site in the Polyoma virus enhancer [18] and has subsequently been implicated in the regulation of genes involved in invasion and metastasis, including collagenase and uPA [19, 33-37]. Although we found that the collagenase Ets element or the Polyoma virus Ets element did not effectively compete for binding to the FGF-BP Ets element, this may reflect the binding of another Ets family member to the FGF-BP promoter whose recognition site could be determined by sequences flanking the GGA core [27].

Deletion of the -47 to -33 FGF-BP promoter region also substantially reduces the TPA effects on the FGF-BP promoter. Sequence analysis revealed that a site homologous to the C/EBP13 binding site is centered in this region of the promoter. The factors binding to FGF-BP C/EBPB element, however, are not effectively competed by the C/EBPB site from the p2 IWAFI/CIPI gene promoter, suggesting that transcription of FGF-BP may be mediated by a different C/EBPB-related factor. The published consensus for C/EBPB is TT/GNNGNAAT/G [38] which is identical in 8 positions (underlined) to the site between -48 to -41 differing only in the most 3'nucleotide of the

consensus. In addition, the involvement of C/EBPB in TPA-mediated responses has been shown previously. For instance, induction by phorbol esters has been shown to cause increased C/EBPß synthesis, phosphorylation, and DNA binding to promoters of a number of genes including MDR1 and collagenase 1 [21-23, 39]. Thus, C/EBPB or a family member is involved in the activity of the -47 to -33 element. Like other leucine- zipper family members, C/EBPB acts co-operatively with other transcription factors to modulate the level of gene expression in response to extracellular stimuli. For example, C/EBPB has been shown to associate with Fos/Jun in vitro [40] and can co-operate in vivo to induce expression of the TSG-6 gene in response to IL-1 and TNF-alpha which is mediated through distinct AP-1 and C/EBPB binding sites in the TSG-6 promoter [41]. Similarly, our data show that the C/EBPB consensus element is a major mediator of TPA-induced gene expression of FGF-BP. However, because removal of the C/EBPB site alone does not completely abolish TPA induction, this suggests that like other TPA- induced genes, the C/EBPB site acts in co-operation with other promoter elements.

A novel aspect of TPA regulation of the FGF-BP promoter is the role of the region -57 to -47 between the AP-1 site and the C/EBPB site. Deletion of this region substantially increases the TPA response, implying that this region normally represses the extent of the response to TPA. A point mutation in this region also abrogates repression thus making it unlikely that the effect of the deletion is simply to bring the AP-1 and C/EBPB sites in closer proximity leading to their increased responsiveness to TPA. In fact, the relief of repression obtained with the -58 point mutant is observed in the presence of the C/EBPB deletion suggesting that the repression impacts on the AP-1 element rather than the C/EBPB site. An alternate possibility is that the factor bound to the -57 to-47 site interacts with the general transcription machinery in a manner similar to the NC2 repressor [42]. However, this seems less likely because we observe no increase in basal activity of the promoter after deletion or mutation of the repressor site in comparison to the -118 construct (Figure 7). The -57 to -47 deletion destroys an

AACGTG (-60 to -55) which is a variant of the CACGTG E-box element recognized by a number of helix-loop-helix factors [24]. The -58 mutant changes the AACGTG to AATGTG and would perturb the 5'part of the dimer recognition sequence [24].

However, the wild type sequence alone does not predict which member of the helix- loop-helix family would interact with this site. Interestingly, binding to an AACGTG recognition element has been described in vitro to a homodimer of the ARNT (aryl hydrocarbon receptor nuclear translocator) helix-loop-helix factor [43] and ARNT deficient embryonic stem cells have a defective angiogenesis process [44]. However, it is unclear whether ARNT homodimers interact with promoters in vivo. Alternatively, other helix-loop-helix factors are known to function as transcriptional repressors, such as the Mad family of proteins which bind related E-box sequences during TPA-induced macrophage differentiation [45, 46] and recruit the mSin3-histone deacetylase corepressor complex, leading to a more closed chromatin structure and transcriptional repression [47].

Through gel retardation analysis, we show distinct factor binding to the AP-1 site and to the E-box repressor site. Interestingly, factor binding to both of these sites is increased upon stimulation with TPA. TPA-induced transcription factor binding to E- box elements has been described for a number of different promoters including c-fos [48-50]. The observation that TPA induces factors which both stimulate and limit induction of FGF-BP suggests a mechanism by which transcription of the FGF-BP gene could be tightly regulated and may reflect a level of tissue-specific expression of this gene.

Overall, our data suggest that the TPA induction of the FGF-BP promoter is induced through both ETS/AP-1 site and a C/EBPB site and that the extent of induction is moderated by factors which bind to an E-box repressor element which lies adjacent to the AP-1 site. It is known that TPA also induces the expression of genes involved in proteolytic degradation of the extracellular matrix such as stromelysin, collagenase, and

urokinase plasminogen activator (uPA) [33, 51, 52]. Interestingly, these promoters are regulated by similar transcription factors as those which we show are involved in FGF- BP promoter induction e. g. Ets/AP-1 and C/EBPB. Thus, our data would support the argument that a specific subset of transcription factors may be induced (or derepressed) to specifically stimulate a panel of genes involved in invasion, angiogenesis and metastasis during skin tumor development.

Additional Findings. It has also been determined that EGF induces transcription of FGF-BP mRNA and PPARy (gamma) agonists reduce transcription of FGF-BP mRNA.

Accordingly, one embodiment of the invention is directed to a nucleic acid comprising an FGF-BP promoter sequence, the sequence comprising an effective portion of the sequence specified in SEQ ID Nô : 1 (shown in Figure 5A and also on deposit in GenBank under accession # AF062639) and a target gene. The promoter sequence may be transcriptionally linked to the target gene. Preferably, the target gene is a detectable marker. However, as will be clear to those of skill in the art, the target gene may comprise any other desired gene, such as a cytotoxic gene, a suicide gene, a tumor suppressor gene, a cell cycle gene, or the thymidine kinase gene, which causes cell death in the presence of the drug gancylovir. The effective portion of the sequence may be any desired length, and preferably comprises at least 10 nucleotides. The nucleic acid is preferably ligated to a suitable plasmid vector.

Another embodiment is directed to a method for detecting an agent that inhibits FGF-BP transcription, comprising the steps of delivering to a cell a nucleic acid comprising a FGF-BP promoter sequence, the sequence comprising an effective portion of the sequence specified in SEQ ID NO : 1 and a gene encoding a detectable marker, such that the nucleic acid molecule is transcribed, adding the agent to the cell, and detecting whether or not the agent inhibits transcription of the detectable marker by

detecting the absence or presence of the marker. The absence of the marker indicates inhibition of FGF-BP transcription. Preferably, the cell is a human cell.

Another embodiment is directed to a compound that inhibits transcription of an FGF-BP gene comprising an agent that inhibits FGF-BP promoter function. The agent may be a drug, a factor, a protein, a nucleic acid or a chemical.

Another embodiment is directed to a method for detecting an agent that induces FGF-BP transcription comprising the steps of delivering to a cell a nucleic acid comprising an FGF-BP promoter sequence, the sequence comprising an effective portion of the sequence specified in SEQ ID NO : 1 and a gene encoding a detectable marker, such that the nucleic acid molecule is transcribed, adding the agent to the cell, alone or in combination with another agent, and detecting whether or not the agent induces transcription of the detectable marker by determining the absence or presence of the marker. In this embodiment the presence of the marker indicates FGF-BP transcription promoting activity of the agent. The agent may be a drug, a factor, a protein, a nucleic acid, a chemical or other substance, and may be administered alone, or in combination with other agents.

Another embodiment is directed to a compound that induces transcription of an FGF-BP gene comprising an agent that induces FGF-BP promoter function. The agent may be a drug, a factor, a protein, a nucleic acid or a chemical.

Another embodiment is directed to a therapeutic compound for use in the treatment of diseases characterized by abnormal FGF-BP expression comprising an agent that alters transcription or expression of FGF-BP. This compound may be used to treat diseases such as cancer, multiple sclerosis, and angiogenesis deficiency, and may also be used to promote wound healing. Cancers or conditions which may be treated include colon carcinoma, squamous cell carcinoma, breast cancer, prostate cancer, and other neoplasias or conditions involving abnormal FGF-BP expression. Depending on the application, the agent may induce or inhibit transcription or expression of FGF-BP.

Other embodiments of the invention are directed to an isolated nucleic acid comprising SEQ ID NO : 1, and to isolated and purified nucleic acid sequences comprising all or an effective fragment of SEQ ID NO : 1. These nucleic acids may be sense or antisense, and may be coupled to a detectable moiety or other desired gene.

The nucleic acids may comprise an element which represses transcription. Preferably, the nucleic acids comprise an E Box repressor element, such as, for example, the sequence -55 to -60 nucleotides upstream of the transcription consensus start site of SEQ ID Nô : 1.

Another embodiment is directed to a nucleic acid sequence which hybridizes preferentially to all or a portion of SEQ ID Nô: 1, wherein the nucleic acid sequence is or is complimentary to all or a portion of the nucleotides of the nucleic acid sequence of SEQ ID NO : 1.

Another embodiment is directed to a composition of matter comprising all or a fragment of a nucleic acid derived from an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1. The nucleic acid may be a sense or antisense nucleic acid and may be single stranded or double stranded.

Another embodiment is directed to a method for treating a disease characterized by abnormal FGF-BP expression comprising administering a composition comprising a pharmaceutically effective amount of an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1. The fragment may be functionally linked to a target gene to be delivered to target cells. The abnormal FGF- BP expression may be overexpression or underexpression. Preferably, the nucleic acid is packaged in a viral vector, which may be any suitable vector, including, by way of example, a retroviral, a vaccinia or an adenoviral vector. The composition may be combined with a pharmaceutically acceptable carrier. Diseases which may be treated include, for example, colon carcinoma, squamous cell carcinoma, breast cancer or prostate cancer.

Another embodiment is directed to a diagnostic kit for detecting the presence or absence of FGF-BP transcription comprising all or a portion of an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1. The sequence may be linked to a detectable label that is radioactive or non-radioactive.

Another embodiment of the invention is directed to a nucleic acid probe that hybridizes to a region of the FGF-BP gene.

Another embodiment is directed to a method of treating a neoplastic disorder comprising administering to a patient a composition comprising a pharmaceutically effective amount of an antisense nucleic acid encoding an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1.

Another embodiment of the invention is directed to a vector comprising a constitutively active promoter functionally linked to an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1. Preferably, the nucleic acid comprises at least 10 nucleotides and may be sense or antisense. The vector may be any suitable recombinant viral vector such as, for example, a retroviral, vaccinia or adenoviral vector. Useful promoters include, but are not limited to, RSV, CMV, SV40, ADV and HSV promoters.

Another embodiment is directed to an expression vector comprising an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1 and a sequence comprising a tumor suppressor gene, wherein the nucleic acid is functionally linked to the sequence. The tumor suppressor gene may be any suitable gene, such as p53, Rb or p21.

Another embodiment is directed to a composition for the treatment or prevention of diseases characterized by abnormal angiogenesis comprising a recombinant vector containing an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1 functionally linked to a tumor suppressor gene, such as, for example a gene encoding p53, Rb or p21.

Another embodiment is directed to a composition comprising an therapeutically effective amount of an expression vector comprising a constitutively active promoter and an antisense FGF-BP promoter sequence, wherein the active promoter is functionally linked to the antisense FGF-BP promoter. The composition may further comprise any pharmaceutically acceptable carrier, such as, for example water, alcohol, oil, saccharide, starch, cellulose, fatty acid, lipid or combinations of any such carriers or other inert ingredients.

Another embodiment is directed to a composition comprising an expression vector comprising an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1 and a nucleic acid sequence comprising a tumor suppressor gene, wherein the tumor suppressor gene is functionally linked to the promoter. The composition may further comprise a suitable carrier. Useful tumor suppressor genes include, but are not limited to p53, Rb or p21.

Another embodiment is directed to a method for treating a patient with cancer comprising the steps of administering an effective dose of a composition comprising an expression vector, wherein the vector comprises a constitutively active promoter functionally linked to an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1. The cancer to be treated may be colon carcinoma, squamous cell carcinoma, breast cancer, prostate cancer or any other neoplasias responsive to the composition. The promoter may be any suitable promoter, including, for example, an RSV, CMV, SV40, ADV or HSV promoter. The effective portion may be an antisense sequence. Preferably, the effective portion is at least 10 nucleotides in length.

Another embodiment is directed to a method of treating a patient with cancer comprising the steps of administering an effective dose of a composition comprising an expression vector, wherein the expression vector comprises an isolated and purified nucleic acid sequence comprising all or an effective fragment of SEQ ID NO : 1,

functionally linked to a tumor suppressor gene. The tumor suppressor gene may be any suitable gene, for example, p53, Rb or p21.

The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention.

Examples Example 1. Cell Culture and Northern Analysis. The ME-180 squamous cell carcinoma cell line was obtained from American Type Culture Collection (ATCC ; Rockville, MD). Cells were cultured in improved minimum essential medium (Biofluids Inc. , Rockville, MD) with 10% fetal bovine serum (Life Technologies, Inc.).

ME-180 cells were grown to 80% confluence on 150 mm tissue culture dishes, washed three times in serum-free IMEM, and then treated 16 hours later with 12-O- Tetradecanoylphorbol 13-Acetate (TPA) (SIGMA) in serum-free IMEM. Total RNA was isolated with the RNA STAT-60 method using commercially available reagents and protocols (RNA STAT-60TM, Tel-Test Inc. ; Friendswood, TX). 30 ,ug of total RNA were separated by electrophoresis in 1. 2% formaldehyde agarose gel and then blotted onto nylon membranes (MSI, Westboro, MA). The blots were prehybridized in 6 x SSC (= 0. 9M sodium chloride, 0. 09M sodium citrate (pH 7. 0)), 0. 5% (w/v) SDS, 5 x Denhardt's solution (= 0. 1 % (w/v) Ficoll, 0. 1 % (w/v) polyvinylpyrrolidone, 0. 1 % (w/v) bovine serum albumin, 100 ug/ml sonicated salmon sperm DNA) (Life Technologies, Inc. ) for 4 hr. at 42°C. Hybridization was carried out overnight at 42°Cin the same buffer. After hybridization, blots were washed three times with 2 x SSC and 0. 1% SDS for 10 min at 42C, and finally once with 1 x SSC and 0. 1% SDS for 20 min at 65°C.

Autoradiography was performed using intensifying screens at -70°C. Blots were stripped by boiling 2 x 10 min in 1 x SSC and 0. 1% SDS. Hybridization probes were prepared by random-primed DNA labeling (Amersham) of purified insert fragments from human FGF-BP [2] and human GAPDH (Clontech). The final concentration of the labeled probes was always greater than 106 cpm/ml of hybridization solution.

Quantitation of mRNA levels were performed using a phosphor-Imager (Molecular Dynamics).

Example 2. In Vitro Transcription On Isolated Nuclei. ME-180 cells were grown to 80% confluence treated or not with 10-7 M TPA for 1,4, or 24 hours on 150 mm tissue culture dishes. Cells were washed three times in serum-free IMEM and then treated 16 hr. later with TPA in serum-free IMEM for indicated times. Nuclei from 107 cells for each time point were isolated after incubation in lysis buffer containing 0. 5 % Nonidet P-40 as described [7]. Nuclear transcription assays were performed with [y- 32p] UTP (Amersham) as described [7]. Equal amounts of radioactivity (0. 5-1 X 107 cpm) were hybridized to nitrocellulose filters on which 3 µg of the pRC/FGF-BP vector was immobilized. B- actin was used as an internal control and pRC/CMV as a background control vector. After hybridization for 4 days at 42°C, the filters were washed 4 times with 2 X SSPE, 0. 1 % SDS for 5 min at 25°C and treated for 30 min at 25°C in 2 X SSPE containing 20 ug/ml RNase A. The filters were then washed 4 times for 30 min in 1 X SSPE, 1 % SDS at 65°C. The amount of radioactivity present in each slot was determined using a phophor-Imager after overnight exposure normalized to the control gene 13-action. Autoradiograms were exposed for 1-3 days with intensifying screens. Quantitative data (mean SD) derived from two independent experiments are shown in Figure 3.

Example 3. Primer Extension Primer 1 was designed from the coding region of the human FGF-BP cDNA (5'-GTGAGGCTACAGATCTTC-3') (SEQ ID NO : 4), primer 2 from the FGF-BP 5'UTR (5'-GTTCACCTTGTTCTGAGCACACGG ATCCA-3') (SEQ ID NO : 5), and a control primer for the 1.2kb kanamycin RNA (Promega). 10 pmoles of each primer was labeled with T4 Polynucleotide Kinase (Promega) and 30 pCi [Y-32P]ATP (Amersham) for one hour and labeled primers were purified over a ChromaSpin-10 gel filtration column (Clontech). Total RNA from ME-

180 cells was isolated as described for Northern Analysis. ME-180 mRNA was purified from total RNA over an Oligo-dT cellulose column (Life Technologies, Inc). 100 fmoles of each FGF-BP-specific primer was incubated with or without 7 ug ME-180 mRNA, or control primer with or without 2 ng 1. 2kb kanamycin control RNA (Promega) in the presence of AMV Reverse Transcriptase buffer (50mM Tris-HCl, 8mM MgCl2, 30mM KC1, ImM DTT, pH 8. 5, Boehringer Mannheim) and allowed to anneal for 1 hour at 50°C. Annealed mixtures were then incubated in the presence of 2 mM dNTPs, 50 units RNase Inhibitor (Boehringer Mannheim), and 40 units AMV Reverse Transcriptase (Boehringer Mannheim) for 1 hour at 42°C. Samples were then run on a 6% polyacrylamide sequencing gel along with radiolabeled Hinf I markers and exposed overnight for autoradiography.

Example 4. Cloning of FGF-BP gene promoter. 1. 8 kb of genomic sequence lying upstream of the human FGF-BP gene was isolated from a human genomic library using the PCR-based PromoterFinder DNA Walking Kit (Clontech) according to the manufacturer's recommendations using rTth XL DNA Polymerase (Perkin Elmer).

Gene-specific primers derived from the 5'UTR of the human FGF-BP cDNA were 5'- ACACGGATCCAGTGCAATCC-3' (+91 to +72) (SEQ ID NO : 6) for the primary round of PCR and 5'-GGAGTGAATTGCAGGCTGCAGCTGTGTCAG-3' (+62 to +33) (SEQ ID NO : 7) for secondary PCR. Secondary PCR products from a DraI library (1. 1 kb) and PvuII library (1. 8 kb) were cloned into a TA Cloning Vector pCR2. 1 (Invitrogen), sequenced by automated cycle sequencing (ABI PRISM Dye Terminator Cycle Sequencing, Perkin Elmer), and confirmed to contain contiguous genomic sequence. This sequence has been submitted to Genbank.

Example 5. Plasmid Constructs. Promoter fragments were cloned into the PXP1 promoterless luciferase reporter vector [9]. PCR products from Pvull (-1829/+62) and Dral (-1060/+62) libraries were removed from pCR2. 1 using HindIII and XhoI sites in the pCR2. 1 multiple cloning site, and ligated into the HindIII and Xhol site of PXP 1

to generate pX-1829/+62Luc and pX-1060/+62Luc. Both constructs contained a BamHI site carried over from the pCR2. 1 vector and located 3'of the BP sequence and 5'of the luciferase gene. The 5'promoter deletion constructs pX-118/+62Luc, pX-93/+62Luc, pX-77/+62Luc, pX-67/+62Luc, pX-56/+62Luc and pX-31/+62Luc were generated by PCR from pX-1060/+62 Luc template using upstream primers spanning -118 to -100, - 93 to -76, -77 to -59, -67 to -49, -56 to -35, and -31 to -11, respectively, all containing a 5'-linked BamHI site. Downstream primer was derived from the luciferase gene 5'- CCATCCTCTAGAGGATAGAATGGCGCCGGGCC-3' (SEQ ID NO : 8). PCR was carried out using Taq DNA Polymerase (Boehringer Mannheim). Products were cut with BamHI, gel purified, and cloned into the PXP1 BamHI site. Correct sequence and orientation were verified by dideoxynucleotide chain termination sequencing with a Sequenase kit 2. 0 (U. S. Biochemical).

Internal promoter deletions were generated by PCR-based site-directed mutagenesis. Complimentary overlapping oligonucleotides containing specific promoter deletions were generated as follows : Ets site deletion from -76 to -67 was incorporated into primers spanning -58 to -93, and -85 to -47, AP-1 site deletion from -65 to -58 was incorporated into primers spanning -47 to -83 and -72 to -35, Ets/AP-1 site deletion from -76 to -58 was incorporated into primers spanning -49 to -93 and -85 to -36, C/EBPB site deletion from -47 to -33 was incorporated into primers spanning -24 to -66 and -57 to -11, and deletion from -57 to -47 was incorporated into primers spanning -35 to -75 and -69 to -27. The -58 point mutant was made by incorporating a C to T point mutation at position -58 into primers representing both srands from -70 to -51. For each construct, two separate PCR reactions were carried out with either the BamHI-linked - 118 to -99 upstream primer or the Luciferase-specific downstream primer. The products were separated from excess primers and mixed, denatured and allowed to reanneal.

Amplification of the heteroduplex with overlapping 3'ends was carried out by 3' extension in the absence of primers followed by amplification using outside primers

(BamHI-linked - 118 to -99 primer and Luciferase-specific primer) in a secondary round of PCR. Final PCR products were digested with BamHI, gel purified, and ligated into the PXP1 BamHI site. Correct sequence and orientation were verified by dideoxynucleotide chain termination sequencing with a Sequenase kit 2. 0 (U. S.

Biochemical). To control for TPA effects on vector sequences unrelated to the FGF-BP promoter insert, we tested a number of non-TPA responsive promoters inserted into the PXP 1 vector for their response to TPA under the conditions used in our experiments : Control vectors, as seen in Figure 6, consisted of fragments of the pro- opiomelanocortin (POMC) promoter [10], the thymidine kinase minimal promoter [9] or the CMV minimal promoter which were cloned into PXP1 vector. All these vectors demonstrated an approximately 2-fold induction after TPA treatment (see Results section).

Example 6 Transient Transfections and Reporter Gene Assavs. 24 hours before transfection, ME-180 cells were plated in 6 well plates in IMEM, 10% FBS at a density of 750, 000 cells/well. For each transfection, 1. 0 ug FGF-BP-luciferase construct and 10p1 Lipofectamine Reagent (Gibco-BRL) were combined in 200R1 IMEM and liposome-DNA complexes were allowed to form at room temperature for 30 minutes.

Volume was increased to lml with IMEM, added to rinsed cells, and incubated for 3 hours at 37°C. Cells were washed and incubated in IMEM for 3 hours then treated for 18 hours with vehicle alone (DMSO, final concentration 0. 1%) or 10-7M TPA.

Transfection efficiency for each construct was determined by co-transfection with 1. Ong of a CMV-driven Renilla luciferase reporter vector pRL-CMV (Promega) and found to be the same for all BP-PXP1 constructs. However, due to a 2-fold background TPA induction of pRL-CMV (see above), results were normalized for protein content and not for Renilla luciferase activity. Cells were lysed by scraping into 150p1 of Passive Lysis Buffer (Promega) and cell debris was removed by brief centrifugation. 20p1 of extract was assayed for both firefly and Renilla luciferase activity using the Dual-

LuciferaseTM Reporter Assay System (Promega). Light intensity was measured in a Monolight 2010 luminometer. Light Units are expressed firefly light units/ug protein.

Protein content of cell extracts was determined by Bradford Assay (Bio-Rad).

Example 7 Gel Shift Assays. ME-180 cells were grown to 80% confluency on 150 mm dishes, serum-starved for 6 hours and treated with or without 10-7M TPA for 90 min. Nuclear extracts were prepared according to Dignam et al. [11] with the following modifications. Pelleted cells were resuspended in 1 mL buffer A (15 mM KCI, 1 OmM HEPES pH 7. 6, 2 mM MgCl2, 0. 1 mM EDTA, 1 mM DTT, 0.1 % Nonidet P-40,1 mM Sodium Orthovanadate) [12] with IX CompleteTM Protease Inhibitor Cocktail (Boehringer Mannheim) and incubated on ice for 10 min. Crude nuclei were pelleted at 700 g and resuspended in 50 ul ice cold buffer C (0. 42 M NaCl, 20 mM HEPES pH 7. 9, 25% glycerol, 0. 2 mM EDTA, 0. 5 mM DTT, 1 mM Sodium Orthovanadate, 1X CompleteTM Protease Inhibitor Cocktail) and vortexed at 4°C for 15 min. After centrifugation for 10 minutes at 1000 g, supernatant was used directly in binding assays and stored at -70°C.

4. 8 pmoles of each synthetic double stranded oligonucleotide was labeled with T4 Polynucleotide Kinase (Promega) and 50 p. Ci [y--PTP (Amersham) for 30 min and labeled primers were purified over a G-25 Sephadex column (Boehringer Mannheim). Binding reactions consisted of 2.5,ug (-80/-63 probe) or 5llg ME-180 nuclear extracts, binding buffer (10mM Tris pH7.5, 10mM KCl, 5% Glycerol, O. ImM DTT, 0.1 mM EDTA), and 200ng poly dIdC (-80/-63 and -70/-51 probes) or 1. 0 ng poly dAdT (-55/-30 probe) and incubated for 10 minutes on ice. Unlabeled competitor oligonucleotides were added and incubated for another 10 minutes before adding 20 fmoles of labeled probe. Reactions were carried out 45 minutes on ice and analyzed by 6% polyacrylamide gel electrophoresis in 1X TBE buffer at 80V. The AP-1 consensus sequence was 5'-CTAGTGATGAGTCAGCCGGATC-3'. (SEQ ID NO : 9).

Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. Although the examples presented herein relate to human applications, as will be clear to those of skill in the art, the present invention may be adapted for use in other species. All references cited herein, including all U. S. and foreign patents and patent applications including U. S. provisional patent application Serial No. 60/093, 778, entitled"Use of FGF-BP Gene Promoter Sequences as Sensors of Drug Effects and Oncogenic, Angiogenic, Vascular, and Immune Function,"filed July 23,1998, are specifically and entirely incorporated by reference. The specification and examples should be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.

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