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Departments of Immunology (X.X., D.M.S.) and Urology (Z.L., K.M.S.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. David M. Spencer, Baylor College of Medicine, One Baylor Plaza/M929, Houston, Texas 77030. E-mail: dspencer{at}bcm.tmc.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Several small animal, noninvasive, high resolution imaging technologies are concurrently under development (reviewed in Ref. 7) that would be potentially useful for in vivo prostate imaging. These technologies vary with respect to several key parameters, including sensitivity, depth penetration, spatial and temporal resolution, throughput, and expense. The highest resolution (25–100 µm spatial resolution) approach, magnetic resonance imaging, based on the differential behavior of electrons in various tissues, suffers from high cost, long sampling times, and low sensitivity in detection of molecular probes. Other relatively high resolution (1–2 mm) and penetrating techniques, such as positron emission tomography and single-photon emission computed tomography, are based on high energy ionizing particles (
-rays) and are, thus, also more expensive to perform than lower resolution (2–5 mm) approaches based on optical bioluminescence or fluorescence. Moreover, these lower cost techniques can provide very high sensitivity and higher throughput. For detecting light emitted from the body, both optical imaging approaches benefit from advances in silicon-based, charge-coupled device (CCD) detectors highly sensitive to visible light (400–1000 nm). Further, newer cooled, CCD cameras can reduce background dark current to almost undetectable levels. Combined with short acquisition times of 10–120 sec (typically) and simultaneous multiple animal imaging capability, high throughput is possible.
The main reported biophysical drawbacks of these two techniques are relative depth sensitivity and low spatial resolution (8). Light intensity drops by an estimated approximately 10-fold for every 1 cm tissue traversed (9), but this is influenced by the tissue type and the degree of tissue vascularization, because light-absorbing hemoglobin proteins are the main detriments to transillumination. As longer wavelength light transilluminates best through biological tissues, red-shifted fluorescent proteins or luciferase (Luc; Ref. 9A ) may decrease the degradation of detectable signal generated intracorporeally but imaged extracorporally. Three-dimensional imaging may further help diminish the impact of these limitations by capturing signal in an arc, much like current computed tomography scanners, or by integration of multiple views of the same animal.
An additional challenge for successful optical imaging is the requirement for exquisite organ- and disease-specific expression of photon-emitting reporter proteins to limit bioluminescence to the tissue of interest. Given the high sensitivity of these imaging techniques, which can potentially detect single cells (or femtomoles of nontissue-specific background reporter expression) (7, 10), current prostate-targeting strategies, which may have appeared adequately prostate specific, have not been previously confirmed at this higher level of discrimination.
To take advantage of the convenience and relative affordability of optical imaging techniques, we have developed transgenic mice with prostate-targeted firefly Luc and enhanced green fluorescent protein (EGFP) using the composite human kallikrein 2 (hK2)-based promoter, hK2-E3/P (11). Kallikreins are the largest subgroup of serine proteases and are often to be found up-regulated in cancer (12). The entire human kallikrein gene locus is present on chromosome 19q13.4 and contains a cluster of 15 tandem genes, called KLK1 to KLK15 (13, 14). Like the prostate-specific antigen (PSA) gene (KLK3), the hK2 gene (KLK2) is up-regulated by androgen in prostate epithelia cells (15). Moreover, PSA and hK2 have served as biomarkers for prostate cancer (16). Compared with PSA, however, hK2 expression has been observed in all assayed prostate cancer cells, including PSA-negative cells, with incremental increases from benign epithelium to high grade prostate intraepithelial neoplasia to adenocarcinoma (17, 18, 19), and with the greatest levels of expression observed in metastatic disease deposits (20). Therefore, we speculated that the hK2 transcriptional regulatory elements might be most useful for the panoply of models, including advanced, metastatic prostate cancer.
Our previous studies demonstrated that the hk2-E3/P promoter is androgen regulated and highly active in androgen receptor-positive (AR+) LNCaP xenografts (11). In the present study we investigated further the tissue specificity of this promoter while developing a novel reporter model for prostate disease. Based on ex vivo measurements, reporter expression is primarily prostate directed, with minor (
5%) signals emanating from the testes and cecum. In living animals the prostate signal is easily visualized; however, the minor signals are also visualized due to the more surface proximal localization of these tissues. Castration reduces the prostate-localized signal by about 50-fold, although in intact mice this decrease is attenuated by confounding intestinal bioluminescence. In contrast, cecum expression was not sensitive to androgen withdrawal due to surgical castration and was also present at similar low levels in female mice. These data demonstrate that noninvasive imaging of relatively small, surface distal tissues is practical and convenient using CCD-mediated detection of bioluminescence reporters. The utility of the EZC model will not only be confined to earlier detection of prostate tumor progression, but should also help to identify metastasis earlier without the intensive effort of a full necropsy and to develop novel antiandrogen receptor reagents.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). For initial validation of androgen dependence, we transiently transfected phK2-E3/P-Luc-IRES-EGFP or control pCMV-EGFP into AR+ LNCaP cells and AR- PC-3 and 293 cells. When cells were transfected with pCMV-EGFP, fluorescence was detected in all three cell lines independent of R1881 stimulation (Fig. 1B and data not shown). However, in phK2-E3/P-Luc-IRES-EGFP-transfected cells, fluorescence was only apparent in LNCaP cells in the presence of R1881. To further determine whether Luc activity is also AR dependent in this construct, the same cells were transiently cotransfected with internal control plasmid, pRL-CMV, and either phK2-E3/P-Luc-IRES-EGFP or ubiquitously expressed pSV40-Luc. Thereafter, cell lysates were prepared, and Luc activity was measured. All pSV40-Luc-transfected cells expressed Luc regardless of androgen stimulation (Fig. 1C). In contrast, when transfected with phK2-E3/P-Luc-IRES-EGFP, Luc activity was only detectable in LNCaP cells in the presence of R1881, but not PC-3 and 293 cells, consistent with the profile of EGFP expression. Moreover, the activity of hK2-E3/P was 6-fold higher than that of the strong SV40 promoter/enhancer in R1881-stimulated LNCaP cells, further confirming the robust strength and AR dependence of this promoter.
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from line 903 and others not shown). All lines, however, displayed similar tissue specificity. Despite relatively high AR levels, very low to negligible EGFP was detected in the testes by this approach. Further, EGFP was undetectable in the other organs tested. As line 903 was the brightest, this line was selected for further development.
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from line 903 and others not shown), consistent with EGFP data. Nevertheless, due to photon dispersion from relatively deep organs (21), it was not possible to determine the source of the detected light. Moreover, a subset of mice from all lines revealed an additional region of light on the left side of the lower abdomen above the predicted location of the prostate. To more unambiguously determine the source of the light, we killed at least five transgenic males immediately after living imaging, dissected their major organs, and placed isolated organs on nonchemiluminescent black paper to be ex vivo imaged. As shown in Fig. 3B, the strongest photon-emitting organ by far was the prostate (ventral prostate showing); however, when the background threshold was set at 1% of the maximal counts per pixel, weak signals could be detected in the testes and cecum. When reset to 3% of the maximum, only prostate-derived light was detectable (not shown). Quantitative analysis with Living Imaging software showed that the light intensity in photon counts from prostate, testis, and cecum was 4.5 ± 0.4 x 106, 8.5 ± 5 x 103, and 2.5 ± 0.4 x 105, respectively. It should be noted that photon signals from the DLP (in the prone position) were much weaker than those from the VP. Obviously, no photon signals originated from other organs, as shown in Fig. 3B. Due to the relative ease of prostate detection in vivo and the reasonably strict prostate specificity of the hK2-E3/P promoter, we renamed line 903 the EZC-prostate model.
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and data not shown). Immunohistochemical analysis using a rabbit anti-Luc antibody demonstrated that the Luc expression was specifically localized to the epithelial cells of the VP and DLP (Fig. 4A). Weak, but reproducible, immunoreactivity was also detected in the testis and cecum. In agreement with the previous images of dissected organs, the staining intensity in the VP was strongest, that in the DLP was less, and those in the testis and cecum were weakest. As before, no positive immunoreactivity was detectable in other organs from transgenic males or from any organs of wild-type males (Fig. 4B and data not shown). Taken together, we demonstrate that the hK2-E3/P promoter is largely specific for the ventral and dorsolateral prostate lobes, with weak expression in the testes and intestines (primarily the cecum).
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). As before, additional signals appeared over the predicted position of the cecum. Given that visible light is significantly (probably exponentially) dispersed and attenuated by intervening tissue, we considered the probability that the observed signal might not accurately reflect the true level of reporter activity in each tissue. Therefore, we killed four males each at multiple (i.e. 3, 6, 12, 24, and 48 wk) time points and removed 27 organs/tissues [i.e. individual prostate lobes [DLP, VP, and anterior prostate (AP)], SV, testis, epididymis, penis, preputial gland, bladder, brain, salivary glands, tongue, thymus, lung, heart, liver, spleen, pancreas, kidney, adrenal gland, stomach, jejunum/ileum, cecum, rectum, bone, muscle, and skin] to quantify Luc enzymatic activity per milligram of tissue extract using a luminometer (Fig. 5B and data not shown). Interestingly, Luc expression was largely restricted to the VP and DLP and increased with age, reaching a peak at 24 wk of age. The significant drop in VP reporter expression at 48 wk probably reflects age-related declines in AR signaling. Only very low relative levels of Luc were detected in the testes (1:62, 1:72, 1:86, and 1:31 at age 6, 12, 24, and 48 wk, respectively) and cecum (1:48, 1:28, 1:33, and 1:1822) compared with that in the VP. The unexpected drop in cecum Luc activity at 48 wk (Fig. 5B) is unclear, but reproducible, despite the apparent high level signal in intact mice (Fig. 5A). Luc activity was not detectable in any other organs tested (data not shown). These results are consistent with those achieved by molecular imaging of living mice or by immunohistochemical analysis; however, weak signals from the cecum and testes are overrepresented relative to signals from the deeper and smaller prostate glands. Thus, in vivo imaging of the prostate is feasible, but adjustments should be made to account for the depth of the prostate tissue. Mechanical manipulation of the testes (into the scrotum) and opaque shielding of the cecum can further improve resolution (not shown).
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, A and B). Photon intensity decreased within 2 d postcastration and remained at reduced levels. At 6 d postsurgery, mice were imaged a final time and immediately killed, and the urogenital tract (including the bladder, seminal vesicles, and prostate) and cecum were removed and arranged for ex vivo imaging. The more quantitative ex vivo images showed that the photon intensity of the intact prostate of castrated mice decreased by 88% (4.5 x 104 vs. 3.8 x 105) compared with that of sham-operated transgenic males (Fig. 6C). Furthermore, analysis of Luc activity per weight using a luminometer demonstrated that the levels of Luc activity actually decreased by 97.2% in the VP and by 97.1% in the DLP castrated animals compared with those in sham-operated controls. In contrast, cecum-derived Luc activity decreased by only 17% in photon intensity when assayed ex vivo (Fig. 6C and data not shown) and by 19.2% in Luc activity measured with a luminometer. Although the full extent of changes in reporter activity is attenuated by light scatter and tissue absorption, these data provide further confirmation that the hK2-E3/P transcriptional element targets transgene expression to the VP and DLP in an androgen-dependent manner.
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). Furthermore, the level of Luc activity in the cecum decreased much less than that in the prostate after castration, suggesting that hK2-E3/P promoter activity in the cecum might be largely androgen independent. To further test this hypothesis, we imaged three transgenic females as before (Fig. 7A). Although the light emission was considerably weaker, a light spot could be captured over the lower abdomen and the region corresponding to the cecum. As before, we killed these mice and reimaged dissected organs ex vivo for more accurate quantitation of transgene expression (Fig. 7B). As in males, photon signals from the cecum were detectable. A very low level of bioluminescence was also visible over the female urogenital tract, and this was confirmed by a standard Luc assay of dissected tissues (Fig. 7C). Furthermore, no Luc activity was detected in any other organ of transgenic females. The levels of Luc activity in the cecum were similar in transgenic males and females (2.5 ± 0.4 x 105 vs. 1.2 ± 0.3 x 105) at 12 wk of age. The slightly higher levels in the male cecum might reflect minor androgen-regulated signaling.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The low level reporter expression in the testes was not surprising, as multiple members of the kallikrein family are androgen regulated and found in the testes (25); however, low level expression in the intestines was not expected. Although unexpected, these findings were consistently observed in six of six lines, suggesting that they were not the result of idiosyncratic transgene integration sites. Although the presence of tissue kallikrein (hK1) is particularly high during inflammatory diseases of the intestines (26), because hK2 is not as good at cleaving kininogen (25), leading to the release of the proinflammatory molecule, lysyl-bradykinin, it is less likely to be found in the inflamed intestines. Nevertheless, weak, but common, transcriptional elements may be shared by multiple kallikrein members.
Using the IVIS CCD system, the in vivo and ex vivo images demonstrated not only that Luc expression can be detected at low levels, but also that all transgenic organs can be rapidly and conveniently screened (Fig. 3), in contrast to conventional, tedious histological methods of screening. In particular, temporal and castration analyses confirmed that hK2-E3/P activity is up-regulated by androgen (Fig. 5) after sexual maturity and down-regulated after castration (Fig. 6) and possibly with aging (Fig. 5). Prostate epithelial specificity was also confirmed by both fluorescence microscopy for EGFP and using a standard luminometer for Luc expression. As hK2-E3/P is a sensitive reporter of AR signaling and not just AR, testosterone, or dihydrotestosterone levels, these reporter mice should permit direct measurement of AR activity under normal and disease states, such as pathogen invasion, aging, genetic disorders, or drug treatment.
Firefly Luc is an enzyme that emits light in the presence of oxygen and its substrate, D-luciferin, and noninvasive bioluminescence imaging technology has been previously used for real-time, low light imaging of gene expression in cell cultures, individual cells, whole organisms, and transgenic organisms (7, 27, 28). The merits of in vivo bioluminescence imaging of transgenic animals have been well documented (7). For example, Lee et al. (29) identified regions of the lactase gene involved in mediating its spatio-temporal expression pattern in the proximal and middle small intestines of developing transgenic mice. Zhang et al. (30) investigated the mechanisms controlling bone morphogenetic protein 4 expression during primordial and mature tissue development (31) and the effects of metalloporphyrins on the transcription of the HO-1 gene in an attempt to target production of neonatal bilirubin. Carlsen et al. (32) has developed transgenic mice that express Luc under the control of the nuclear factor-
B promoter, enabling real-time imaging of nuclear factor-B activity and its modulation in living mice. Most recently, Ciana et al. (21) demonstrated that estrogen receptors are active in immature transgenic mice before gonadal production of sex hormones as well as in ovariectomized adult transgenic mice. These transgenic studies provide insight into the tissue-specific and developmental activities of various promoters, and they give valuable insight into the potential utility of those promoters for therapeutic application.
Although a powerful new tool in animal modeling of development and disease, tissue-specific transcriptional regulatory elements have been widely suggested as reagents to increase the safety and effectiveness of gene therapy (33, 34, 35, 36). However, the activity of the majority of these promoters is much weaker than that of commonly used, nontissue-specific, virus-based promoters, such as the CMV promoter, leading to poor efficacy (37, 38). To solve this problem, one creative strategy has been to employ a two-step transcriptional amplification system to amplify the expression of Luc and HSV1-sr39tk in a prostate cancer cell line using a composite PSA-based promoter/enhancer to express GAL4 derivatives fused to VP16 activation domains. The resulting activators were targeted to cells with reporter templates bearing GAL4-binding, upstream activation sites (39, 40). Although that approach shows enhanced efficacy, the benefits are somewhat tempered by the logistics of a binary approach. In contrast, the triplicate enhancer/promoter, hK2-E3/P, was comparable to the CMV enhancer/promoter in AR-positive LNCaP cells, but was inactive in the AR-negative prostate cancer line PC-3 and in all nonprostate cancer lines tested.
Although we noted in this study that in vivo imaging is quite convenient, Luc-based bioluminescence measurements using the IVIS system are currently significantly biased by signal depth due to light scattering and absorption. Further, commercially available Luc enzymes, such as beetle Luc from the North American firefly (Photinus pyralis) or coral Luc from the sea pansy (Renilla reniformis), oxidize their substrates, D-luciferin or coelenterazine, respectively, to produce primarily green (
max, 562 nm) or blue (480 nm) light, respectively light (41, 42). Although these wavelengths are easily detectable with a sensitive CCD system, light intensity drops by approximately 1 log/cm tissue or more due to light attenuation by living tissue (9). This high signal attenuation probably explains the large discrepancy between signals seen by imaging intact and castrated mice in vivo compared with luminescence in tissue preparations (Fig. 6). The use of mutant Luc enzyme emitting red-shifted light upon luciferin oxidation should greatly minimize these discrepancies in the future, and development of these new models is underway (43).
Also, in intact mice it is not possible to determine the exact source of light emission (Figs. 3 and 5–7). Three-dimensional resolution could be improved by strategic placement of perpendicular detectors or mirrors. Because the prostate is very small and is localized in a relative deep position, imaging the murine prostate in intact mice is especially challenging, compared with, for example, that in the testes or cecum. Nevertheless, as long as the hK2-E3/P promoter stays active in neoplastic tissue, analogous to human prostate adenocarcinoma, prostate-restricted growth, observed in hyperplastic or neoplastic autochthonous models, should be easy to quantify, as signal changes should be spatially restricted. Moreover, imaging of distal metastasis in bone or soft tissue (e.g. lungs and liver) should be straightforward. Breeding to the transgenic adenocarcinoma of mouse prostate autochthonous tumor model (6) and to the inducible fibroblast growth factor receptor 1 model, JOCK-1 (3), are currently underway to test this hypothesis. Finally, the development of reporter models based on even more tissue-specific promoters is well underway along with the design and development of conditional reporter models that will stay on constitutively once activated regardless of androgen levels.
In conclusion, we confirmed that the hK2-E3/P promoter is a potent, largely prostate-specific and androgen-dependent promoter in a transgenic model using bioluminescence imaging technology. Although better models are being developed, this model should provide a convenient new tool to assess changes in the size of the prostate after transformation, progression, and drug treatment.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Constructs
Composite promoter, hK2-E3/P, was released from pSH1/hK2-E3/P-SEAP (11) by SacII/XbaI digestion and inserted into the SacII and XbaI sites of pKBPA (44) to obtain phK2-E3/P-KBPA. The resulting plasmid was digested with EcoRI, Klenow-blunted, and annealed to the blunted ClaI/SmaI fragment of pBS-Luc-IRES-EGFP (11) to give phK2-E3/P-Luc-IRES-EGFP. pCMV-EGFP was previously described (11). Plasmids pSV40-Luc, containing firefly Luc gene under the transcriptional control of the SV40 enhancer/promoter, and pRL-CMV, containing the Renilla Luc reporter gene, were obtained from Promega Corp. (Madison, WI).
Transfections
Cells were seeded in six-well plates at 40–50% confluence at 37 C (5% CO2) in complete RPMI 1640 medium containing 10% charcoal/dextran-treated FBS, 18 h before transfection. Cells were transfected with plasmid DNA using FuGENE 6 (Roche, Indianapolis, IN) by the manufacturer’s method. The ratio of plasmid DNA to FuGENE was optimized for each cell type and varied from 1–2 µg DNA to 3 µl FuGENE. To compare the activities of transcriptional regulatory elements with each other, cells were cotransfected with phK2-E3/P-Luc-IRES-EGFP and pRL-CMV expressing the Renilla Luc gene as an internal control. Unmodified expression vector, pSH1, was used as a negative control, and pSV40.E/P-Luc was used as a positive control. After a 5-h incubation, synthetic androgen (methyltrienolone/R1881, NEN/DuPont, Boston, MA) was added for AR stimulation.
Generation and Screening of Transgenic Mice
The minimal eukaryotic-derived fragment of hK2-E3/P-Luc-IRES-EGFP was released from plasmid phK2-E3/P-Luc-IRES-EGFP with ClaI/SalI restriction enzymes (Invitrogen) and gel-purified using the Gene Clean Kit (Bio 101, Vista, CA). The hK2-E3/P-Luc-IRES-EGFP transgenic mice were generated by microinjection of the purified fragments at 10 ng/µl into the FVB male pronuclei of fertilized oocytes by the transgenic core laboratory at Baylor College of Medicine. Mice were maintained in the Transgenic Mouse Facility, a pathogen-free environment, in compliance with Baylor College of Medicine policy. Mouse tail DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, CA) and was screened by PCR using primers for the firefly Luc gene: Luc-P1, 5'-AGCCAGCATGGAAGACGCCAAAAAC-3'; and Luc-P2, 5'-ATCGCAGTATCCGGAATGATTTGATTGC-3'. The DNA quality control primers for mouse casein were: forward, 5'-GATGTGCTCCAGGCTAAAGTT-3'; and reverse, 5'-AGAAACGGAATGTTGTGGAGT-3'.
Luciferase Assays
Transiently transfected cells were lysed and assayed for Luc activity using the Dual-Luciferase Reporter Assay System (Promega Corp.) following the manufacturer’s protocol with a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). The fold (over control, formula: (b/a) x (c/d), where a is firefly Luc units of control, b is firefly Luc units of sample, c is Renilla Luc units of control, and d is Renilla Luc units of sample) of Luc activity was expressed as the mean of triplicate transfections, which were repeated at least three times.
To assay tissue-derived Luc activity, animals were euthanized and dissected. Tissue specimens from the VP, DLP, and AP lobes were microdissected and stored temporarily on ice before homogenization in lysis buffer. Other tissue specimens, including testis, epididymis, SV, and penis (from males); uterus, ovary, and oviduct (from females); as well as brain, salivary glands, thymus, lung, heart, stomach, cecum, liver, adrenal gland, kidney, muscle, skin, and bone, were also analyzed to determine the tissue specificity of the hK2-E3/P promoter. Tissues were homogenized with a PRO250 homogenizer (Pro Scientific, Inc., Oxford, CT) in 300 µl Luc lysis buffer (Promega) containing 1:100 diluted protein inhibitor cocktail (Roche). Specimens were centrifuged at 8000 rpm for 5 min and placed temporarily on ice. The Luc activity of the cell lysates was measured with a TD 20/20 luminometer (Turner Designs, Inc., Mountain View, CA), and the protein concentration was determined using the detergent-compatible protein assay system (Bio-Rad Laboratories, Hercules, CA) in a DU-640 spectrophotometer (Beckman Instruments, Fullerton, CA). The luminescence results are reported as relative light units per milligram of protein.
Specific Expression of EGFP in Vitro and in Vivo by Fluorescence Microscopy
Cells were transferred to six-well plates at 50% confluence in complete medium containing 10% charcoal/dextran-treated FBS 16 h before transfection. Cells were transfected with phK2-E3/P-Luc-IRES-EGFP or pCMV-EGFP, using FuGENE 6 (Roche). Forty-eight hours postinfection, cells were photographed with a digital fluorescence microscope system. The tissue samples indicated above for Luc assays were also frozen-sectioned for determination of EGFP expression.
Histological and Immunohistochemical Examinations
The microdissected prostate parts and other tissues indicated above were fixed in 1% paraformaldehyde for 4 h and transferred to 70% ethanol overnight. Tissues were processed in a series of increasing ethanol concentrations and embedded in paraffin wax. Five-micrometer sections were cut and stained with hematoxylin and eosin. Immunostaining for firefly Luc was also performed. Briefly, sections were deparaffinized in xylene, rehydrated in decreasing alcohol from 100% to 80%, and microwaved with 10 mM citrate buffer (pH 6.0) at 95–99 C for 10 min for antigen retrieval. Finally, endogenous peroxidase activity was quenched with 3% hydrogen peroxide. Nonspecific binding was abolished with 10% Power Blocker (BioGenex, San Ramon, CA) for 10 min. The tissue sections were incubated with a 1:2000 dilution of 10 mg/ml goat antifirefly Luc antibody conjugated with biotin (Abcam, Hartford, CT) overnight at 4 C. After PBS washes with 0.1% Tween 20 for 2 h, sections were incubated with horseradish peroxidase-conjugated streptavidin using the Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA) for 45 min at room temperature. Peroxidase activity was determined with 3',3'-diaminobenzidine tetrahydrochlorate using a 3',3'-diaminobenzidine kit (Vector Laboratories, Inc.), according to the protocol provided. Finally, sections were washed in distilled water for terminating the reaction, counterstained with 1% methyl green, dehydrated, and mounted.
Imaging and Quantification of Bioluminescence Data
Mice were anesthetized with a mixture of oxygen/isoflurane using an Inhalation Anesthesia System (VetEquip, Inc., Pleasant Hill, CA). D-Luciferin (Molecular Probes, Eugene, OR) was ip injected at 25 mg/kg mouse body weight. Ten minutes after D-luciferin injection, mice were imaged with an IVIS Imaging System (Xenogen, Alameda, CA), consisting of a cooled CCD camera mounted on a light-tight specimen chamber (dark box), a camera controller, a camera cooling system, and a Windows-based computer system. Imaging parameters were maintained for comparative analysis. Gray scale-reflected images and bioluminescence-colorized images were superimposed and analyzed using the Living Image software (Xenogen). A region of interest (ROI) was manually selected over relevant regions of signal intensity. The area of the ROI was kept constant, and the intensity was recorded as maximum photon counts within an ROI (41). In some experiments, after imaging living mice, animals were euthanized, and organs of interest were removed, arranged on black, bioluminescence-free paper, and ex vivo imaged within 30 min.
Castration of Mice
To study the effect of androgen ablation on hK2-E3/P-driven firefly Luc expression, male mice, aged 12 wk, were anesthetized with avertin (2,2,2-tribromoethenol, Sigma-Aldrich Corp., St. Louis, MO) at 240 mg/kg mouse weight, and cleaned around the scrotum with 70% ethanol. A 1-cm median incision was made at the tip of the scrotum. The testes lying in the sacs can be seen by placing pressure on the lower abdomen. A 5-mm incision was made in each sac, and the testis, epididymis, vas deferens, and spermatic blood vessels were pulled out. A single ligature was placed around the spermatic blood vessels and vas deferens. The testis and epididymis were removed by severing the blood vessels and vas deferens distal to the ligature. The remaining vas deferens was pushed back into the sac, and the incision was sutured. The castrated mice were put on a heating pad until recovery. Sham-operated, age-matched males were used as controls. The orchiectomized mice were imaged every other day until d 6 postcastration, when animals were euthanized and imaged ex vivo as described above.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AP, Anterior prostate; AR, androgen receptor; BPH, benign prostatic hyperplasia; CCD, charge-coupled device; CMV, cytomegalovirus; DLP, dorsolateral prostate; E, enhancer; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; hK2/KLK, human glandular kallikrein protein and gene; Luc, luciferase; P, promoter; PIN, prostatic intraepithelial neoplasia; PSA, prostate-specific antigen; R1881, methyltrienolone; ROI, region of interest; SV, seminal vesicle; SV40, simian virus 40; VP, ventral prostate.
Received for publication August 19, 2003. Accepted for publication December 12, 2003.
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B activity. J Immunol 168:1441–1446This article has been cited by other articles:
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