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Experimental Therapeutics |
Departments of Radiation Medicine [I. T., A. Di., M. J., A. Dr., V. S.] and Biochemistry and Molecular Biology [D. R., M. S.], Georgetown University Medical Center, Washington, DC 20007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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40,000 deaths annually in the United States. Whereas >80% of the tumors initially respond to androgen ablation, metastatic prostatic cancer inevitably progresses to an androgen-independent state. Once this happens, the disease is difficult to control, because hormonally independent tumors become resistant to additional hormonal manipulations as well as chemotherapy or radiotherapy (1)
. PARP3 is a zinc finger-containing protein, allowing the enzyme to bind either double- or single-strand DNA breaks. Numerous studies have shown that PARP is involved in a variety of biological functions, all of which are associated with breaking and rejoining of DNA strands, and plays a pivotal role in DNA damage repair (reviewed in Refs. 2, 3, 4 ). Recent advances in apoptosis research have identified PARP as one of the intracellular "death substrates," and have demonstrated that limited proteolysis of PARP by caspases is an early event or perhaps a prerequisite for the execution of programmed cell death in a variety of cells (5) . The caspase-specific DEVD motif resides adjacent to the nuclear localization signal of the PARP protein. Cleavage of PARP at this site results in the separation of the two zinc finger DNA-binding motif in the NH2 terminus of PARP from the automodification and catalytic domains located in the COOH terminus of the enzyme. Consequently, this cleavage excludes the catalytic domain from being recruited to the sites of DNA fragmentation during apoptosis and presumably disables PARP from coordinating subsequent repair of genome maintenance events. The biological function of the DNA-binding domain of PARP has been investigated by using stable cell lines expressing PARP-DBD protein (6) . Data obtained from these experiments indicate that PARP-DBD expression in mammalian cells: (a) leads to trans-dominant inhibition of PARP; (b) has no effect on normal cell proliferation; and (c) sensitizes the cells to genotoxic agents and ionizing radiation. Exposure of the PARP-DBD-expressing cells to these DNA-damaging agents results in a marked reduction of cell survival, increased frequency of sister chromatid exchanges, inhibition of cell proliferation, and apoptosis induction (7) . Thus, the DBD of PARP offers a potential for targeted sensitization of tumor cells to genotoxic agents and radiotherapy.
In the past few years several new approaches for treating advanced neoplasms have been proposed, including that of gene therapy. Differential expression of the desired gene product in the target tissue is central to the concept of gene therapy. One such approach is to use tissue-specific promoters to drive therapeutic genes. From this point of view, the promoter of the gene encoding the PSA represents a promising tool for prostate cancer-specific gene expression (8) . Although low levels of PSA are detectable in the serum of men with normal prostates, PSA expression is increased in most patients with prostate cancer, regardless of tumor stage and hormone responsiveness. The promoter of the PSA gene has been cloned, and its two functional domains have been identified: a proximal promoter and a distal promoter, which can also function as an enhancer (9) . Using LNCaP tumor xenografts in the nude mouse model it was demonstrated that the PSA promoter retained its tissue-specific properties in vivo (10) . Furthermore, the PSA promoter was able to mimic the prostate-specific and androgen-regulated expression of the PSA gene in transgenic mice (11) . Thus, the PSA promoter contains the features that are fundamental for the development of expression vectors for prostate-specific gene therapy: tissue specificity and androgen responsiveness.
The present study reports a novel approach for combination therapy that uses the tissue-specific (prostate) and DNA damage-specific (targeting the PARP function) gene therapy for prostate cancer. We describe the development of recombinant plasmids for expression of the DNA-binding domain of PARP under control of prostate tissue-specific promoter in PSA producing LNCaP prostate carcinoma cells. Our results show that enforced PARP-DBD expression significantly augments sensitivity of these cells to DNA-damaging treatments, presenting a novel strategy for gene therapy directed to prostate cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Plasmid Constructs.
The 1.3-kb fragment that contains the upstream enhancer element (9
, 10)
of the PSA regulatory region (nt -745 to -2080) was cloned from human placenta using a PCR-generated probe corresponding to nts 1–200 of PSA cDNA. The PSA gene minimal promoter (nt -619 to +12) was obtained by performing PCR amplification using human placenta genomic DNA as a template, and set of primers 5'-GGTCTGGAGAACAAGGAGTG (upstream) and 5'-TCTCCGGGTGCAGGTGGTAA (downstream) designed according to the reported sequence data for the 5' region of the human PSA gene (9)
. The 0.7-kb fragment of the human PARP cDNA encoding for DNA binding domain (aa 1–234) was PCR amplified using plasmid pCD12 containing cDNA for human PARP as a template and primers designed according to the reported sequence data (12)
. The human cDNA coding for the DNA-binding domain of PARP (5'-EcoRI-HindIII) was inserted into pcDNA 3.1 (-) expression vector at the EcoRI/HindIII restriction sites downstream of the human CMV promoter/enhancer. Subsequently, PARP-DBD was tagged at its COOH terminus with a sequence encoding four Flag-epitope tags (13)
. The resulting recombinant plasmid, pCMV-DBD/F, permits constitutive expression of human PARP-DBD under control of the CMV promoter. To express the human PARP-DBD under control of the PSA gene regulatory elements, the CMV promoter sequences were replaced with a 1336-bp XhoI-EcoRV fragment of PSA enhancer fused with an EcoRI fragment containing 662-bp sequence of PSA promoter. The resulting plasmid, pPSA(e/p)-DBD/F is designed to express human PARP-DBD in androgen-inducible and PSA-dependent fashion.
Transient DNA Transfections.
DNA transfections were carried out using an activated-dendrimer (Superfect; Qiagen) as described (13)
. Cells (2 x 105) were transfected with 5 µg of pCMV-DBD/F or pPSA(e/p)-DBD/F plasmids using a ratio of DNA to Superfect reagent of 1:10. Assays for PARP-DBD expression were performed 48 h after transfection.
Stably Transfected LNCaP Cell Lines.
Cells were transfected with pPSA(e/p)-DBD/F, pCMV-DBD/F, or with control, neomycin-resistant expression vector p
CMV-DBD/F, respectively, using Superfect reagent (Qiagen). The G418-resistant colonies from each replicated experiment were pooled to form polyclonal cell populations and were routinely maintained in medium containing 300 µg/ml G418.
PARP-DBD Immunodetection.
Logarithmically growing cells were washed twice with cold PBS and lysed at 4°C for 30 min in buffer: 0.5% Triton X-100, 0.5% NP40, 2 mM NaOV4, 150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 20 µg/ml leupeptin. For immunoprecipitation, cell lysates were normalized for protein content and incubated with anti-Flag M2 monoclonal antibody agarose affinity gel (Sigma), followed by Western blotting using polyclonal anti-PARP antibody (R&D System; 1:1000) directed against the aa 71–329 of PARP protein. The secondary antibody was donkey antigoat IgG conjugated to horseradish peroxidase (Santa Cruz, 1:2000). Signals were detected using the ECL system (Amersham). For in situ PARP-DBD immunodetection, LNCaP cells (PSA-DBD) were grown on poly-D-lysine-treated glass slides (Fisher Scientific). After induction of PARP-DBD expression by synthetic androgen R1881 (10 nM) for 24 h, cells were subjected to fixation with 3.7% paraformaldehyde and incubated for 30 min with anti-Flag M2 monoclonal antibody (Sigma; dilution 1:200). Washes were followed by 30-min incubation with Cy-5 conjugated secondary antibody (Jackson ImmunoResearch; dilution 1:200) in PBS, contained 10% donkey serum and 0.1% 300 Bloomgelatin. Transmitted and Cy5 fluorescence images were acquired using an IX 70 confocal microscope (Olympus, Melville, NY).
DNA Binding Assays.
PARP and PARP-DBD affinity for DNA was assayed using double-stranded oligonucleotides coated onto magnetic beads. Briefly, 100 µg of streptavidin-coated Dynabeads (Dynal Biotech) were incubated with 120 pmols of 5'-biotinylated double-stranded pJ
(5'-GTGAAAAAGGTGAAAAAG) oligonucleotides (14)
in accordance with the manufacturer’s instruction. Crude cell extracts in IP buffer were prepared from cells transiently transfected with pCMV-DBD/F or from parental LNCaP cells, and were normalized for protein content. Purified PARP protein (Alexis; specific activity 30 units/µg) or cell lysates were combined with pJ-affinity beads and incubated for 30 min with gentle agitation at room temperature. The protein-bound beads were separated using a magnetic separator (Dynal), and bound proteins were eluted with 30 µl of 1 M NaCl and subsequently analyzed by Western blotting using goat polyclonal anti-PARP antibody (R&D Systems). Recombinant human PARP and streptavidin-coated beads containing no DNA were used as positive and negative controls, respectively.
RT-PCR Analyses.
RNA was isolated from cells using TRIzol Reagent (Life Technologies, Inc.) according to the manufacturer’s protocol. The primers for human DBD-Flag fusion protein were: sense, 5'-ATCACCATCACCATCA-3' and antisense, 5'-CCTTTATCGTCATCGT-3'. RT-PCR was performed using 2 mg of total cellular RNA and the ThermoScript RT-PCR System (Life Technologies, Inc.).
Proliferation and Apoptosis Assays.
Growth characteristics of LNCaP cells were assayed by a colorimetric method using the tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (Cell Titer 96 Aqueous Assay; Promega). Surface expression of phosphatidyl serine was determined by FITC-labeled annexin V staining (Trevigen) followed by analysis using a fluorescence-activated cell sorting Star Plus flow cytometer (Becton Dickinson). Apoptotic cells were defined as FITC positive and propidium iodide negative. Changes in the mitochondrial potential were analyzed using JC-1 DePsipher (Trevigen) reagent as suggested by manufacturer. Stained samples were analyzed at 488 nm argon laser by flow cytometry.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. The recombinant plasmid, pCMV-DBD/F, permits constitutive expression of the PARP-DBD under control of the human CMV promoter. To achieve tissue-specific expression of the PARP-DBD in the androgen-sensitive LNCaP cells, we have constructed an expression vector, pPSA(e/p)-DBD/F, comprised of the coding region for the DNA-binding domain of PARP linked to 5'-flanked sequences (1.3 kb upstream enhancer and 0.6 kb minimal promoter) of the human PSA gene. The expression of the PARP-DBD Flag-fusion protein in LNCaP cells was confirmed in transient transfection assays (Fig. 1B)
. Immunoblot analysis of cell lysates revealed that exogenous PARP-DBD Flag fusion protein has a molecular mass of 31 kDa consistent with the length of corresponding cDNA, and is recognized by anti-Flag and anti-PARP antibodies. Functional activity of expressed PARP-DBD Flag-fusion protein was assayed in DNA binding reactions using double-stranded 5'-biotinylated oligonucleotides coupled to streptavidin-coated magnetic beads. These beads were used to recover DNA-binding proteins from LNCaP cells transiently transfected with pCMV-DBD/F plasmid. We found (Fig. 1C)
that both endogenous PARP and PARP-DBD fusion protein are captured effectively by DNA fragments, thus indicating that PARP-DBD retain its DNA-binding activity when expressed in LNCaP prostate carcinoma cells.
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The tissue specificity of PARP-DBD expression under control of the PSA promoter/enhancer was evaluated in transient transfection assays using the PSA-producing (LNCaP) and PSA-negative (PC-3) prostate cancer cells, as well as cells of nonprostate origin such as Ewing’s sarcoma (A4573 cell line). We found that PSA enhancer/promoter-driven expression of the human PARP-DBD was immunodetectable only in PSA-producing LNCaP prostate carcinoma cells but not in PSA-independent cell lines (Fig. 2)
. Although more PSA-producing cell lines need to be tested to elaborate a PSA dependence of PARP-DBD expression, our data are consistent with findings reported previously that PSA promoter retains its tissue specificity both in vivo and in vitro (11
, 15)
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. Parental LNCaP cells and the LNCaP cell subline (CMV-DBD) were used as negative and positive controls, respectively, for PARP-DBD expression levels in these experiments. We found that exposure of PSA-DBD cells to androgen (R1881) resulted in dose-dependent stimulation of PARP-DBD expression at levels of mRNA (Fig. 3B)
and protein (Fig. 3A)
. No notable changes in the PARP-DBD expression levels have been observed in control cell lines exposed to R1881 at doses up to 10 nM (data not shown). Androgen-dependent regulation of PARP-DBD expression in PSA-DBD prostate carcinoma cells was additionally confirmed by in situ immunodetection of the PARP-DBD-Flag fusion protein using fluorescence microscopy (Fig. 3C)
. These data indicate that the pPSA(e/p)-DBD/F recombinant vector allows expression of functionally active DBD of PARP in vitro and that the androgen-dependent expression is specific to PSA-producing prostate carcinoma cells.
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To investigate whether PARP-DBD would increase the susceptibility of human prostate carcinoma to DNA-damaging treatments, the expression of PARP-DBD in LNCaP (PSA-DBD) cells was induced by R1881, and cells were subsequently exposed to ionizing radiation or etoposide (VP-16). We found that androgen (R1881) -dependent stimulation of PARP-DBD expression significantly enhanced (at least 2-fold) growth inhibition of PSA-DBD cells in response to DNA damage, compared with control cells (Fig. 4A)
. This inhibition can be attributed to PARP-DBD expression in LNCaP cells (Fig. 3)
rather then to the presence of androgen in the growth medium. In fact, other studies have shown that androgens are potent stimulators of LNCaP cells growth in vitro (17)
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. When PARP-DBD expression was induced by R1881, irradiated or etoposide-treated LNCaP (PSA-DBD) cells showed significantly (>2-fold) increased staining for annexin V (Fig. 4B)
and depolarization of mitochondrial membrane (Fig. 4C)
within 24 h of treatment. These data indicate that perturbation of PARP function via enforced expression of its dominant-negative mutant (PARP-DBD) results in enhanced sensitivity of prostate cancer cells to DNA-damaging treatments. Considering the fact that androgens block apoptosis in LNCaP cells triggered by diverse agents, including ionizing radiation (19)
, our observations suggest that overexpression of the PARP-DBD augments apoptotic pathways in these cells in an androgen-independent fashion. Additional investigations are required to elucidate the mechanisms for PARP-DBD-mediated sensitization of LNCaP cells to DNA damage, as well as the enhanced apoptotic responses in DBD-expressing prostate cancer cells. The studies addressing these questions are currently underway. Although many prostate cancer cells are deficient in DNA mismatch repair, they are resistant to ionizing radiation and DNA-damaging drugs. Therefore, targeting molecular components that are critically involved in maintenance of genome stability is a promising approach directed at overcoming intrinsic tumor cell resistance to DNA-damaging treatments. From this point of view, the strategy described here represents a novel starting point for the design of PARP-based molecular therapies targeting prostate cancer in vivo. First, this approach uses tissue-specific (prostate carcinoma) and treatment-specific (DNA damage) gene therapy for prostate cancer. Next, to avoid the potential side effects due to expression of PSA in tissues other than prostate, tumor cells are targeted using an agent that is not functionally active in the absence of massive DNA damage and, therefore, would not be toxic to cells outside of the irradiated volume or pose a genetic risk to the patient. Furthermore, PARP-DBD-mediated cell death is independent of cell proliferation states because both nondividing cells and rapidly proliferating cancer cells cannot survive the massive accumulation of long-lived damage in the genome (20) . Thus, targeting tumor cells with the PARP-DBD can be beneficial especially for the control of prostate cancer, because prostate cancers usually grow very slow. These properties of the PARP-DBD are in marked contrast to conventional chemotherapeutic drugs, which primarily target proliferating cells. In summary, the plasmid vector developed in this study permits the expression of the human PARP-DBD in an androgen-inducible and PSA-dependent fashion, and sensitizes prostatic adenocarcinoma cells to DNA-damaging treatments. These results provide a proof-of-principle for a novel therapeutic strategy to control prostate cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by grants from the United States Army Medical Research and Development Command DAMD 17-00-1-0019 and DAMD 17-00-1-0276 (to V. S.) and the National Cancer Institute PO1 CA 74145 (to A. Dr. and M. S.). Fluorescent microscopy and flow cytometry analyzes were performed using the Shared Resources of the Lombardi Cancer Center, Georgetown University, Washington, D.C. (USPHS Grant 2P30-CA-51008). 
2 To whom requests for reprints should be addressed, at Department of Radiation Medicine, Georgetown University, The Research Building, Room E-204A, 3970 Reservoir Road, N.W., Washington, DC 20007. Fax: (202) 687-0400; E-mail: soldates{at}georgetown.edu
3 The abbreviations used are: PARP, poly(ADP-ribose) polymerase; PARP-DBD, DNA binding domain of poly(ADP-ribose) polymerase; PSA, prostate-specific antigen; nt, nucleotide; aa, amino acid; RT-PCR, reverse transcription-PCR.
Received 6/ 4/02. Accepted 9/26/02.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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) or absence (
) of synthetic androgen R1881 (10 nM) before irradiation (20 Gy) or treatment with etoposide (10 µM). Viable cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium assay at indicated times, and results are expressed as a percentage of mock-treated control (n = 4); bars, ±SD. B, effect of PARP-DBD expression on annexin staining in LNCaP cells after DNA-damaging treatments. PSA-DBD cells were maintained in absence or in presence of R1881 (10 nM) for 24 h before irradiation (IR; 20 Gy) or treatment with etoposide (10 µM; VP-16). Annexin V binding activity was determined in parental LNCaP and PSA-DBD cells by flow cytometry 24 h after treatments. Apoptotic cells are defined as annexin V-positive cells and are expressed as percentage of total cell number in sample analyzed on FACSscan flow cytometer. Data presented are the mean values determined from triplicate experiments; bars, ±SD. C, effect of PARP-DBD expression on changes of mitochondrial membrane potential in LNCaP cells after DNA-damaging treatments. After treatments (24 h), untreated controls (UT), irradiated (IR; 20 Gy), or etoposide-treated (10 µM; VP-16) cells were stained with JC-1 "DePsipher" reagent and analyzed by flow cytometry. Mitochondrial potential breakdown in dying cells results in accumulation of green fluorescent JC-1 monomers, which, in turn, is reflected by an increase of green fluorescence events. Representative data (of three independent experiments) are shown.