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Experimental Therapeutics |
Calydon, Inc., Sunnyvale, California 94089
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Insertion of the hK2 enhancer/promoter into adenovirus to drive the E1A genes of adenovirus type 5 (Ad5) created an attenuated replication competent adenovirus variant Calydon virus (CV) 763, which replicates similarly to wild-type adenovirus in prostate tumor cells but is attenuated in nonprostate tumor cells. In addition, CV764, an adenovirus variant containing the previously cloned prostate-specific enhancer (to drive the Ad5 E1A genes) and the hK2 enhancer/promoter (to drive the Ad5 E1B genes) was constructed. CV764 is significantly attenuated and has a high therapeutic index with a cell specificity of 10,000:1 for PSA(+) LNCaP cells, compared to ovarian cancer OVCAR-3 cells and SK-OV-3 cells and PA-1 cells. CV764 is also highly attenuated in primary human microvascular endothelial cells.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PSA is a well-characterized and important marker for prostate cancer (7 , 8) . hK2 protein is also becoming an important marker for prostate cancer. With the development of monoclonal antibodies directed against hK2 protein, several studies have found that hK2 protein circulates in different relative proportions to PSA in the blood of patients with prostate carcinoma (9, 10, 11) . It was also noted that a number of PSA-negative tumors had detectable hK2 protein (10) . More importantly, hK2 protein was recently shown to be expressed in every prostate cancer, and the expression of hK2 protein incrementally increased from benign epithelium to high-grade prostatic intraepithelial neoplasia to adenocarcinoma. In contrast, PSA and prostate acid phosphatase displayed inverse concentrations compared to hK2 protein with disease progression (12) . These data suggest that hK2 protein may add new information to prostate cancer diagnosis, characterization, and progression. More recently, the hK2 protein was demonstrated to be involved in the activation or maturation of PSA. Both the PSA and hK2 proteins are synthesized as propeptides that are cleaved in the prostate to yield the mature forms found in semen. The zymogen form of PSA, which has no or very low enzyme activity, is activated after incubation with enzymatic quantities of mature hK2 protein (13, 14, 15, 16) .
The hK2 promoter has been shown to contain a functional ARE that is inducible in prostate cells (17) . Data produced from PSA studies indicate that a single ARE is insufficient to drive androgen induction and that upstream sequences containing a second ARE must participate in the induction process (18, 19, 20, 21) . It is, therefore, important to identify and characterize those potential cis elements and trans activators of the hK2 gene to obtain more insight into the mechanism of androgen action in the prostate.
Here, we report that a 12.3-kb fragment upstream of the hK2 gene contains an enhancer with an ARE that is selectively active in LNCaP cells in vitro. This enhancer stimulates a 50–100-fold increase in transcription when linked to the hK2 promoter. In addition, a potential therapeutic, CV764, an ARCA containing the PSE, driving the Ad5 E1A genes, and the hK2 TRE, driving the Ad5 E1B genes, was constructed. CV764 is genetically stable and replicates with a preference toward prostate cancer cells expressing PSA of as much as 10,000:1.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning of hK2 5'-Flanking Region and Plasmid Construction.
The DNA region between the first exon in the hK2 gene and the 3' end of the PSA gene was amplified from human genomic DNA (Boehringer-Mannheim) by PCR with primers 42.100.1 and 42.100.4. 42.100.1 is complementary to the 5'-UTR of the first exon in the hK2 gene (6)
. 42.100.4 corresponds to the 3'-UTR of the PSA mRNA. The resulting PCR fragment was ligated into pGEM-T vector (Promega) to create the plasmid CP312. pGL3-Basic, pGL3-Promoter and pGEM-T vectors were purchased from Promega Corp. pCMV ß-gal was purchased from Clontech. CP296 contains the hK2 fragment from -2394 to +33, relative to the transcription start site, which was amplified from CP312 by PCR with primers 42.100.1 and 42.100.3. CP300 was constructed by inserting the hK2 fragment from -2394 to +33 released from CP296 by NcoI-SacI digestion and ligated into a similarly cut pGL3-Basic in wild-type orientation. CP325 has the hK2 minimal promoter (-324 to +33) driving the luc coding region. The minimal hK2 promoter was amplified from genomic DNA with two primers, 42.100.1 and 43.121.2. To further narrow down the fragment, a series of plasmids were generated by PCR with a number of synthetic oligonucleotides. CP377 is a luc reporter construct containing a hK2 5' flanking region amplified by PCR with CP312 as template and two synthetic oligonucleotides 51.70.1 and 51.70.2. The PCR product was digested with KpnI and XhoI, ligated into a similarly cut CP325, creating CP377. Similarly, CP378, CP379, CP380, CP383, CP387, CP390, CP396, and CP412 were generated by the same procedure using the primers 51.70.3, 51.70.4, 51.70.6, 51.96.1, 51.96.2, 51.96.3, 51.96.4, and 51.116.2, respectively.
Generation of ARE Mutations.
Mutations were introduced into the AREII of CP390 by using the Quickchange site-directed mutagenesis kit (Stratagene). Two different sets of primers introduced 6-bp substitutions in the same six locations in the AREII. The mutations introduced by primers 58.104.1 and 58.104.2 created plasmid CP457. The mutations introduced by primers 58.104.3 and 58.104.4 created plasmid CP458. Both of these plasmids are the same as CP390, with the exception of the mutations in the AREII. Mutants were confirmed by DNA sequencing.
DNA Transfection and luc Assays.
For transfection, LNCaP cells were plated at 5 x 105 cells per 6-cm culture dish in complete medium. Reporter constructs were introduced into the cells as described previously (18)
. When more than one construct was used, 5 µg of the smallest construct were used, and proportionally larger amounts of DNA were used for each other construct to keep the plasmid molar concentrations equal and constant; exceptions are noted. After a 4-h incubation with the DNA complexes, the culture medium was aspirated and replaced by RPMI 1640 containing 10% charcoal/dextran-treated fetal bovine serum (stripped serum) supplemented with 10-9M methyltrienolone (R1881; DuPont/NEN) or the appropriate hormone. After an additional 48-h incubation at 37°C/5% CO2, cells were harvested in 500 µl of lysate buffer (Analytical Luminescence Laboratories). Fifty µl of this lysate were used to measure luc activity in a microtiter plate luminometer (ML3000; Dynatech Laboratories). luc activities were corrected for variation in protein concentrations of the cell extracts (Bio-Rad Protein Assay) or normalized to ß-gal activity (Tropix). luc activities and relative induction factors were expressed as means from at least three independent experiments.
Virus Construction and Characterization.
pXC.1 and pBHG10 were purchased from Microbix, Inc., and were described previously (22)
. To generate E1A and E1B ARCA mutants, a platform plasmid (CN124) with two unique restriction sites was constructed from pXC.1. pXC.1 contains the left-end human Ad5 sequence from bp 22 to 5790. CN124 contains an AgeI site at bp 547 between the E1A mRNA cap site and E1A translation initiation site and an EagI site at 1681 between the E1B promoter and E1B mRNA cap site. The AgeI site was created by inserting a thymidine between Ad5 bp 551 and 552. yielding CN95 (23)
. The EagI site was created by inserting a guanine nucleotide between Ad5 bp 1681 and 1682 using overlap PCR with the following two sets of primers. The first set, 15.133A and 9.4, amplifies a 2090-bp fragment in CN95, and the second set, 9.3 and 24.020, amplifies a 399-bp fragment from the same plasmid. CP306 is a derivative of CP124 in which a 68-nucleotide fragment upstream of the E1A cap site was deleted to remove the endogenous Ad5 E1A promoter. CP421 was constructed by cloning the hK2 enhancer domain from -5155 to -3387 relative to the transcription start site and the promoter from -324 to +33, into the PinAI site of CP306. The hK2 enhancer/promoter was amplified by PCR from CP379 with two synthetic primers 51.116.3 and 42.100.1, and the PCR product was digested with PinAI and ligated into a similarly cut CP306, creating CP421. CP416 is a plasmid in which the E1A gene driven by PSE and the E1B gene is driven by hK2 enhancer/promoter. First, the PSE fragment was amplified from CN706 DNA with primers 51.10.1 and 51.10.2, digested with PinAI and ligated into similarly cut CP306, producing CP321. Second, the hK2 enhancer/promoter fragment was amplified from CP379 with primers 42.174.2 and 51.116.6, digested with EagI, and ligated into a similarly cut CP321, creating CP416.
Adenovirus variant CN702 contains a wild-type E1 region and an E3 deletion, whereas CN706 has the PSE driving the E1A gene and the identical E3 deletion, as described previously (23) . ARCA variants CV763 and CV764 were constructed with the identical E3 deletions as CN702 and CN706 by cotransfecting plasmids CP421 or CP416 DNA and BHG10, as described previously (23) . Viral constructs were confirmed by Southern blotting. Two experiments were conducted to characterize the growth properties of the adenovirus variants. For the virus yield (in pfu/cell) assay, 2 x 105 293, LNCaP, HBL-100, OVCAR-3, SK-OV-3, and PA-1 cells were plated in duplicate into six-well plates. Twenty-four h later, medium was aspirated and replaced with 0.5 ml of serum-free RPMI 1640 containing the indicated adenovirus at a MOI of 2 pfu/cell. After a 2-h incubation at 37°C, cells were washed twice with prewarmed PBS, and 2 ml of complete RPMI 1640 were added to each well. After an additional 48 h, the cells were scraped into the culture medium, and the cells were lysed by three freeze-thaw cycles. The supernatant of each duplicate point was tested for virus production by triplicate plaque assay for 10–12 days under semisolid agarose on 293 cells (24 , 25) . For the cytopathic effect assay, primary hMVEC cells were grown to 80% confluence and infected with either CV764 or CN702 for 2 h at increasing MOI from 0.01 to 10. Plates were monitored daily for cytopathic effect, and the assay was terminated when essentially total cytolysis was observed at a MOI of 0.1 with CN702.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 42.100.1 is complementary to the 5'-UTR of the first exon in the hK2 gene (6)
. 42.100.4 corresponds to the 3'-UTR of the PSA mRNA. Both strands of the 12.3-kb region between the PSA and hK2 genes were sequenced by fluorescent dye terminator labeling using AmpliTaq DNA polymerase. The PCR product was verified by overlapping sequence identity to published sequences at the 5' flanking region of the hK2 and 400 bp at the 3' flanking region of the PSA gene. In addition, the 12.3-kb PCR product had the same pattern as genomic DNA by Southern blotting (data not shown). The 12.3-kb sequence has been deposited with GenBank accession number AF113169.
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summarizes the data, showing that CP383 is highly inducible by the synthetic testosterone analogue R1881. At 1.0 nM, CP383 was 
25-fold more active than CP325. At 0.0 nM R1881, CP325 was 22-fold over background (pGL3-Basic-transfected LNCaP cells), and CP383 was 3-fold more active than CP325; see Fig. 1
). These data suggest that the 7.3-kb 5'-flanking sequence contains an androgen-responsive transcriptional regulatory element.
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). In particular, the sequence between -3819 to -3805 in the minus strand matches the consensus ARE at 13 of 15 positions (26, 27, 28)
. Glucocorticoid response half-sites were also identified at -4296 and -3797 in the plus strand and -3549 in the minus strand. Additional factor binding sites identified included a seven of seven match to the CREB consensus at -2843, a six of seven match to the AP-1 binding site at -4076, and a six of seven match to the c-Fos serum response element at -4412. There is high homology (66%) between the hK2 5'-flanking region from -6000 to +8 and that of the PSE. Alignment analysis of the sequence found 75% homology between the PSE (-5322 to -3738; Ref. 18
) and the far upstream region of hK2 (-5155 to -3387; Fig. 5
). Upon further sequence analysis, the ARE sequence of hK2 gene between -3819 and -3805 (5'-GGAACATATTGTATT-3') is identical to the AREII (-4148 to -4134) found in the PSE (Refs. 18
and 21
; Fig. 5
).
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. The deletion of the sequences between -2394 and -324 (CP377) did not lead to any loss of androgen inducibility comparing with CP383. The same is true if the sequence between -7020 to -5155 was deleted (CP378). Removal of the sequence between -3387 to -2394 (CP379) or -3643 to -2394 (CP387) resulted in a 70–90-fold induction of luc activity. When the deletion from 3' border was extended to include half of the putative AREII sequence (CP380), the level of induction was approximately the same as that seen with CP325. Further deletion at 5' border was examined. A construct containing the hK2 5'-flanking region from -3643 to -4814 (CP390) showed the highest inducibility, a 102-fold increase. CP396 was 30-fold inducible. When the deletion from 5' border was extended to -3884 (CP412), the gain in induction was abolished. The combination of these results indicates that the putative AREII sequence as well as its upstream region are required for high level hK2 expression. These data also suggest that a fragment of the human glandular kallikrein regulator, from -4814 to -3643 is very active in the up-regulation of hK2, whereas the sequence located from -3993 to -3643 still maintains significant activity. In addition, these data suggest negative regulatory sequences also exist. For example, CP390 has more activity than CP387, which has more activity than CP379, which, in turn, has more activity than CP377; these results suggest negative regulatory elements between -5155 and -4814, -3643 and -3387, and -3387 and -2394, respectively.
Characterization of the hK2 Regulatory Element.
To determine whether the regulator of hK2 gene has enhancer properties, constructs containing the regulatory element from CP379 (-5155 to -3387) inserted in various positions and orientations relative to the hK2 promoter/luc gene transcription unit were generated. Transient transfection showed that this regulatory element stimulated transcription regardless of its location or orientation relative to the promoter upon which it acts (data not shown), indicating that the hK2 regulatory element is an enhancer.
The results of previous experiments suggest that the hK2 promoter responds in an androgen-dependent manner (17)
. By using an SV40 promoter, we found that the hK2 enhancer can also contribute to androgen responsiveness and is, thus, independent of the promoter used (data not shown). This is similar to the enhancer of the PSA gene (18)
. To further test whether the putative ARE in this enhancer region was functionally active, the sequence was mutated to 5'-GtActATATTacAgT-3' (CP457) or to 5'-GcAgaATATTcgAAt-3' (CP458). In transfection experiments, the mutated enhancer was no longer active or R1881 inducible (Fig. 2A)
. These results suggest that the putative ARE (AREII) is functionally active and also provides evidence that AREII plays a pivotal role in the androgen regulation of hK2.
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. The androgens tested, included a naturally occurring androgen, DHT, and a synthetic androgen, methyltrienolone (R1881). Both androgens caused increases in luc activity when compared to cells with no hormone treatment. However, LNCaP cells treated with DHT did not show an increase similar to those found with R1881 treatment. This may be due to differences in their affinity for the androgen receptor (28)
or to the fact that R1881 is not metabolized by LNCaP cells, whereas the half-life of DHT in LNCaP cells supplemented with stripped serum medium is 30 min.4
Nonandrogenic compounds, including DES, a synthetic estrogen, and DEX, a synthetic glucocorticoid, showed little or no inducibility (Fig. 2B)
. Thus, the hK2 enhancer and promoter confer androgen-mediated gene transcription.
Tissue Specificity of the hK2 Gene Enhancer.
The hK2 protein is recognized to be a potentially useful biomarker of prostate cancer and is predominately expressed in the prostate gland (10
, 11)
. Tissues outside of the prostate synthesize very little or no hK2 protein. It was, therefore, important to determine whether the enhancer described above retained not only androgen responsiveness, but also the high level of tissue specificity characteristic of the hK2 gene.
To test tissue specificity, we transfected a variety of cell lines with three reporter constructs: CP325 (-324 to +33), CP390 (-4814 to -3643 and -324 to +33), and CP396 (-3993 to -3643 and -324 to +33). The cell lines used represent several hormone-responsive tissues including human breast epithelia (HBL-100), human breast carcinoma (MCF-7), colon carcinoma (LoVo), liver carcinoma (HUH-7), lung carcinoma (A549), ovary carcinoma (OVCAR-3), and prostate carcinoma (LNCaP, androgen receptor positive; PC-3, androgen receptor negative). The 293 cell line was derived from human embryonic kidney cells transformed by the E1 region of Ad5. The cell lines were transfected with reporter DNAs and an internal control plasmid, pCMVß-gal (cytomegalovirus promoter driving the ß-gal gene). The summary of results is shown in Fig. 3
. In LNCaP cells, CP390 and CP396 stimulated 100- and 35-fold increases, respectively, in luc production in the presence of 1 nM R1881, whereas CP325 stimulated a 6-fold increase of luc. In no other cell line did CP390 or CP396 lead to a >6-fold induction of luc synthesis. The highest levels of activity outside of LNCaP cells were observed in MCF-7, where CP325 and CP390 reached 1.6- and 1.5-fold, respectively. Not surprisingly, all three hK2 reporter constructs were inactive in the PC-3 prostatic carcinoma cell line because it lacks a functional androgen receptor (27, 28, 29, 30)
. These data suggest that the hK2 enhancer retains a high level of tissue specificity and requires an androgen receptor for activation.
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. CV763 contains the hK2 TRE from the enhancer domain (-5155 to -3387) and the proximal promoter (-324 to +33) cloned at the E1A transcription start site to drive E1A expression. CV764 contains a copy of the PSE sequence (23)
at the E1A transcription start site to drive E1A expression and a copy of hK2 TRE at the E1B transcription start site to drive E1B expression. CN702, an adenovirus that has a wild-type E1 region, was used as a wild-type control in this study. CN706 contains a copy of PSE at E1A transcription start site to drive E1A expression and showed selective cytotoxicity toward PSA-expressing cells in vitro and in vivo (23)
. Virus structures were confirmed by PCR and Southern blotting and found to be genetically stable (data not shown).
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indicate that the viruses CN702, CN706, CV763, and CV764 equally produced 1 x 104 pfu per cell in 293 cells. Slightly lower yields were found with the PSA(+) LNCaP cells in which CN702 produced 5 x 103 pfu/cell, whereas CN706, CV763, and CV764 equally produced 1–2 x 103 pfu/cell. However, the prostate specific ARCA variants CN706, CV763, and CV764 grew poorly on nonprostate cells. For example, CV763 and CN706 yielded 100-fold less virus/cell in HBL-100, OVCAR-3, SK-OV-3, and PA-1 cells than the wild-type E1 virus CN702. This indicates that the hK2 TRE engineered adenovirus preferentially replicates in prostate tumor cells. Of greater interest, CV764, a virus with PSE driving E1A and hK2 TRE driving E1B, is significantly replication restricted in nonprostate tumor cells. The virus yield (in pfu/cell) decreased by 5000-fold in HBL-100 cells, 8000-fold in PA-1 cells, and 10,000-fold in SK-OV-3 and OVCAR-3 cells when compared to CN702. Indeed, CV764 yielded <1 pfu/cell in all of the PSA(-) cells, a rate of replication that clearly cannot sustain an active self-sustaining virus replication.
To characterize the differential viral cytopathic effects in primary human cells, we performed CPE assays. Nonimmortalized hMVECs were chosen to test sensitivity to CV764 and wild-type adenovirus (CN702) infection. As shown in Fig. 4C
, CN702 caused monolayer cytolysis of hMVEC monolayers at MOI as low as 0.01 within 10 days. In contrast, CV764 infected hMVEC monolayers did not show significant cytopathic effects at the same time points with MOI of 10, 1.0, 0.1, and 0.01. Cytolysis of hMVECs with CV764 equivalent to that seen with wild-type CN702 adenovirus was only evident at a MOI 1,000–10,000-fold greater than the MOI used with CN702. Thus, CV764-mediated cytolysis is significantly attenuated relative to wild-type adenovirus in primary normal hMVECs.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As members of the human kallikrein family, the hK2 and PSA genes share a number of characteristics. Both genes are expressed predominately in the male prostate (5
, 31
, 32)
, although minor levels of PSA and hk2 are found in female breast cells (33
, 34)
. Both genes are up-regulated by androgens by transcriptional activation (35
, 36)
. Both hK2 and PSA mRNAs are synthesized predominantly in prostatic epithelia (6
, 31)
. We demonstrate here that the hK2 and PSA genes contain similar regulatory elements (Fig. 5)
. (a) With respect to the cap site, there is a 75% homology between the hK2 enhancer and the PSE in the 5'-flanking region from -5300 to -3300. The hK2 enhancer and the PSE contain an ARE (AREII) at about -4000, which is identical in sequence (18
, 21)
. Additionally, several consensus binding sites for c-Fos, AP-1, and CREB transcription factors were found in the hK2 enhancer and PSE with a similar localization (Fig. 5
and Ref. 18
). (b) Both the hK2 enhancer/promoter and the PSE are androgen responsive and androgen specific. (c) Both the hK2 enhancer/promoter and the PSE stimulate gene expression only in the prostate tumor cells. (d) Both the hK2 enhancer/promoter and the PSE bind in a sequence-dependent manner one or more proteins found in LNCaP cells but not in HeLa cells (18)
.5
Taken together, our data suggest that not only is the expression of the hK2 and the PSA genes regulated by similar control mechanisms but also that the structural and regulatory genes of hK2 and PSA evolved by gene duplication. But why did the hK2 protein evolve to cleave zymogen PSA into active PSA when animals below primates do not even contain a PSA gene (37)
?
We applied the characterization of the hK2 enhancer/promoter to the construction of potential therapeutics. We demonstrate here ARCA variants containing hK2 enhancer/promoter sequences driving early Ad5 genes with restricted replication to prostate tumor cells. CV763 (hK2 enhancer/promoter driving the Ad5 E1A genes) and CV764 (PSE driving the Ad5 E1A genes and hK2 enhancer/promoter driving the Ad5 E1B genes) attenuated virus yield per cell and had no cytopathic effects in nonprostate cells. However, both CV763 and CV764 replicate as well as wild-type adenovirus (CN702) in prostate tumor cells. Interestingly, the virus yield per cell of CV763 is similar in HBL-100 and OVCAR-3 cells to that of CN706, a virus whose E1A gene is under the control of PSA TRE (23) . Yet the virus yield per cell of CV764 was 100-fold less than CN706 or CV763 in these nonpermissive cell types and 5,000–10,000-fold less than CN702.
Importantly, if ARCA variants are to be considered for i.v. treatment of metastatic prostate cancer, the expected virus yield per cell in nontarget cells must be low. Otherwise, a productive virus multiplication cycle could be initiated in nontarget cells. In vitro studies of CV764 reported here show a 5,000–10,000-fold reduction of virus yield (pfu/cell) compared to the wild-type E1 but E3 deleted CN702 virus in breast epithelial HBL-100 cells, ovarian cancer OVCAR-3, SK-OV-3 cells, and PA-1 cells. In each of these cases, the virus yield per cell of CV764 dropped below 1, indicating an inability of CV764 to sustain a productive virus multiplication cycle in these PSA(-) cells. Thus, this study extends the general method of ARCA to construct highly specific cytotoxic agents of increasing utility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank (accession no. AF113169). 
2 To whom requests for reprints should be addressed, at Calydon Inc., 1324 Chesapeake Terrace, Sunnyvale, CA 94089. Phone: (408) 734-0733; Fax: (408) 734-2808; E-mail: dhenderson{at}calydon.com
3 The abbreviations used are: PSA, prostate-specific antigen; ARE, androgen response element; CV, Calydon virus; PSE, PSA enhancer; Ad5, adenovirus type 5; TRE, transcription response element; ARCA, attenuated replication competent adenovirus; hMVEC, human primary microvascular endothelial cell; UTR, untranslated region; ß-gal, ß-galactosidase; CP, Calydon plasmid; luc, luciferase; pfu, plaque-forming unit(s); MOI, multiplicity of infection; CREB, cAMP-responsive element binding protein; AP-1, activator protein-1; DHT, dihydrotestosterone; DES, diethylstilbestrol; DEX, dexamethasone.
4 H. G. Lamparski and D. R. Henderson, unpublished data.
5 D-C. Yu and D. R. Henderson, unpublished data.
Received 8/24/98. Accepted 1/29/99.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 W.-S. Cheng, V. Giandomenico, I. Pastan, and M. Essand Characterization of the Androgen-Regulated Prostate-Specific T Cell Receptor {gamma}-Chain Alternate Reading Frame Protein (TARP) Promoter Endocrinology, August 1, 2003; 144(8): 3433 - 3440. [Abstract] [Full Text] [PDF]
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J. L. Jakubczak, P. Ryan, M. Gorziglia, L. Clarke, L. K. Hawkins, C. Hay, Y. Huang, M. Kaloss, A. Marinov, S. Phipps, et al. An Oncolytic Adenovirus Selective for Retinoblastoma Tumor Suppressor Protein Pathway-defective Tumors: Dependence on E1A, the E2F-1 Promoter, and Viral Replication for Selectivity and Efficacy Cancer Res., April 1, 2003; 63(7): 1490 - 1499. [Abstract] [Full Text] [PDF]
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 M. Messina, D. M. T. Yu, G. W. Both, P. L. Molloy, and B. G. Robinson Calcitonin-Specific Transcription and Splicing Targets Gene-Directed Enzyme Prodrug Therapy to Medullary Thyroid Carcinoma Cells J. Clin. Endocrinol. Metab., March 1, 2003; 88(3): 1310 - 1318. [Abstract] [Full Text] [PDF]
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A. Jain, A. Lam, I. Vivanco, M. F. Carey, and R. E. Reiter Identification of an Androgen-Dependent Enhancer within the Prostate Stem Cell Antigen Gene Mol. Endocrinol., October 1, 2002; 16(10): 2323 - 2337. [Abstract] [Full Text] [PDF]
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D. M. Nettelbeck, A. A. Rivera, C. Balague, R. Alemany, and D. T. Curiel Novel Oncolytic Adenoviruses Targeted to Melanoma: Specific Viral Replication and Cytolysis by Expression of E1A Mutants from the Tyrosinase Enhancer/Promoter Cancer Res., August 15, 2002; 62(16): 4663 - 4670. [Abstract] [Full Text] [PDF]
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J. Zhang, N. Ramesh, Y. Chen, Y. Li, J. Dilley, P. Working, and D.-C. Yu Identification of Human Uroplakin II Promoter and Its Use in the Construction of CG8840, a Urothelium-specific Adenovirus Variant That Eliminates Established Bladder Tumors in Combination with Docetaxel Cancer Res., July 1, 2002; 62(13): 3743 - 3750. [Abstract] [Full Text] [PDF]
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K. A. Rauen, D. Sudilovsky, J. L. Le, K. L. Chew, B. Hann, V. Weinberg, L. D. Schmitt, and F. McCormick Expression of the Coxsackie Adenovirus Receptor in Normal Prostate and in Primary and Metastatic Prostate Carcinoma: Potential Relevance to Gene Therapy Cancer Res., July 1, 2002; 62(13): 3812 - 3818. [Abstract] [Full Text] [PDF]
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C.-L. Hsieh, L. Yang, L. Miao, F. Yeung, C. Kao, H. Yang, H. E. Zhau, and L. W. K. Chung A Novel Targeting Modality to Enhance Adenoviral Replication by Vitamin D3 in Androgen-independent Human Prostate Cancer Cells and Tumors Cancer Res., June 1, 2002; 62(11): 3084 - 3092. [Abstract] [Full Text] [PDF]
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 C. J. A. Ring Cytolytic viruses as potential anti-cancer agents J. Gen. Virol., March 1, 2002; 83(3): 491 - 502. [Abstract] [Full Text] [PDF]
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Y. Li, D.-C. Yu, Y. Chen, P. Amin, H. Zhang, N. Nguyen, and D. R. Henderson A Hepatocellular Carcinoma-specific Adenovirus Variant, CV890, Eliminates Distant Human Liver Tumors in Combination with Doxorubicin Cancer Res., September 1, 2001; 61(17): 6428 - 6436. [Abstract] [Full Text] [PDF]
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X. Xie, X. Zhao, Y. Liu, J. Zhang, R. J. Matusik, K. M. Slawin, and D. M. Spencer Adenovirus-mediated Tissue-targeted Expression of a Caspase-9-based Artificial Death Switch for the Treatment of Prostate Cancer Cancer Res., September 1, 2001; 61(18): 6795 - 6804. [Abstract] [Full Text] [PDF]
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C. Balague, F. Noya, R. Alemany, L. T. Chow, and D. T. Curiel Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes J. Virol., August 15, 2001; 75(16): 7602 - 7611. [Abstract] [Full Text] [PDF]
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S. Matsubara, Y. Wada, T. A. Gardner, M. Egawa, M.-S. Park, C.-L. Hsieh, H. E. Zhau, C. Kao, S. Kamidono, J. Y. Gillenwater, et al. A Conditional Replication-competent Adenoviral Vector, Ad-OC-E1a, to Cotarget Prostate Cancer and Bone Stroma in an Experimental Model of Androgen-independent Prostate Cancer Bone Metastasis Cancer Res., August 1, 2001; 61(16): 6012 - 6019. [Abstract] [Full Text] [PDF]
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Y. Chen, T. DeWeese, J. Dilley, Y. Zhang, Y. Li, N. Ramesh, J. Lee, R. Pennathur-Das, J. Radzyminski, J. Wypych, et al. CV706, a Prostate Cancer-specific Adenovirus Variant, in Combination with Radiotherapy Produces Synergistic Antitumor Efficacy without Increasing Toxicity Cancer Res., July 1, 2001; 61(14): 5453 - 5460. [Abstract] [Full Text] [PDF]
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H. Yamamura, M. Hashio, M. Noguchi, Y. Sugenoya, M. Osakada, N. Hirano, Y. Sasaki, T. Yoden, N. Awata, N. Araki, et al. Identification of the Transcriptional Regulatory Sequences of Human Calponin Promoter and Their Use in Targeting a Conditionally Replicating Herpes Vector to Malignant Human Soft Tissue and Bone Tumors Cancer Res., May 1, 2001; 61(10): 3969 - 3977. [Abstract] [Full Text]
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G. M. Yousef and E. P. Diamandis The New Human Tissue Kallikrein Gene Family: Structure, Function, and Association to Disease Endocr. Rev., April 1, 2001; 22(2): 184 - 204. [Abstract] [Full Text]
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K. Doronin, M. Kuppuswamy, K. Toth, A. E. Tollefson, P. Krajcsi, V. Krougliak, and W. S. M. Wold Tissue-Specific, Tumor-Selective, Replication-Competent Adenovirus Vector for Cancer Gene Therapy J. Virol., April 1, 2001; 75(7): 3314 - 3324. [Abstract] [Full Text]
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M. Brunori, M. Malerba, H. Kashiwazaki, and R. Iggo Replicating Adenoviruses That Target Tumors with Constitutive Activation of the wnt Signaling Pathway J. Virol., March 15, 2001; 75(6): 2857 - 2865. [Abstract] [Full Text]
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D.-C. Yu, Y. Chen, J. Dilley, Y. Li, M. Embry, H. Zhang, N. Nguyen, P. Amin, J. Oh, and D. R. Henderson Antitumor Synergy of CV787, a Prostate Cancer-specific Adenovirus, and Paclitaxel and Docetaxel Cancer Res., January 1, 2001; 61(2): 517 - 525. [Abstract] [Full Text]
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D.-C. Yu, Y. Chen, M. Seng, J. Dilley, and D. R. Henderson The Addition of Adenovirus Type 5 Region E3 Enables Calydon Virus 787 to Eliminate Distant Prostate Tumor Xenografts Cancer Res., September 1, 1999; 59(17): 4200 - 4203. [Abstract] [Full Text] [PDF]
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), and the constructs with an x through the ARE site (CP457 and CP458) are constructs with the ARE mutated as described in "Materials and Methods." The wild-type sequence of the ARE and the mutated sequences are listed beneath each construct. B, steroid specificity of hK2 enhancer activity. LNCaP cells were transfected with 5 µg of CP390. The cells were incubated with one of the following steroids: 50 nM DEX, 50 nM DES, 10 nM DHT, or 1 nM R1881. Columns, fold induction over duplicate identical samples at 0 nM each hormone; bars, SD.
, heterologous transcription regulatory sequences. 
, adenovirus sequence. CV763 contains transcriptional regulatory elements from the hK2 gene (-5155 to -3387 and -324 to +33) engineered at adenovirus genome bp 547 to direct expression of the E1A gene. CV764 contains a copy of the PSE and the hK2 promoter/enhancer engineered to direct expression of both the E1A gene and the E1B gene. CN702 and CN706, a wild-type adenovirus and a prostate-specific ARCA, were described previously