Noninvasive Molecular Imaging of MYC mRNA Expression in Human Breast Cancer Xenografts with a [ 99m...

Noninvasive Molecular Imaging of MYC mRNA Expression inHuman Breast Cancer Xenografts with a [99mTc]Peptide-PeptideNucleic Acid-Peptide Chimera

Xiaobing Tian,† Mohan R. Aruva,‡ Wenyi Qin,| Weizhu Zhu,| Edward R. Sauter,|

Mathew L. Thakur,‡,§ and Eric Wickstrom†,§,*

Departments of Biochemistry and Molecular Pharmacology, Radiology, and Kimmel Cancer Center,Thomas Jefferson University, Philadelphia, Pennsylvania 19107, and Department of Surgery,University of Missouri, Columbia, Missouri 65212. Received August 28, 2004;Revised Manuscript Received October 28, 2004

Human estrogen receptor-positive breast cancer cells typically display elevated levels of Myc proteindue to overexpression of MYC mRNA, and elevated insulin-like growth factor 1 receptor (IGF1R) dueto overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC peptidenucleic acid (PNA) probes with an IGF1 peptide loop on the C-terminus, and a [99mTc]chelator peptideon the N-terminus, could measure levels of MYC mRNA noninvasively in human IGF1R-overexpressingMCF7 breast cancer xenografts in nude mice. We prepared the chelator-MYC PNA-IGF1 peptide, aswell as a 4-nt mismatch PNA control, by solid-phase synthesis. We imaged MCF7 xenograftsscintigraphically and measured the distribution of [99mTc]probes by scintillation counting of dissectedtissues. MCF7 xenografts in nude mice were visualized at 4 and 24 h after tail vein administrationof the [99mTc]PNA probe specific for MYC mRNA, but not with the mismatch control. The [99mTc]-probes distributed normally to the kidneys, livers, tumors, and other tissues. Molecular imaging ofoncogene mRNAs in solid tumors with radiolabel-PNA-peptide chimeras might provide additionalgenetic characterization of preinvasive and invasive breast cancers.

INTRODUCTION

MYC was one of the first proto-oncogenes to be identi-fied (1), and MYC was the first oncogene to be inhibitedby antisense oligonucleotides, blocking the transitionfrom G1 to S phase in normal peripheral blood mono-nuclear cells (2) and in promyelocytic leukemia cells (3).MYC mRNA and Myc protein are overexpressed in avariety of cancers, including breast (4), bladder (5),kidney (6), lung cancer (7), and lymphoma (8). MYCantisense oligonucleotides were able to ablate Myc pro-tein levels in peripheral blood mononuclear cells of Eµ-myc transgenic mice (9), delay the onset of congenitallymphoma in Eµ-myc transgenic mice (10), and delay theproliferation of Eµ-myc lymphoma cells transplanted intocongenic parental mice (11). Similarly, in patients withadvanced solid tumors, a phase I trial of our MYCantisense oligonucleotide sequence, combined with cis-platin, revealed no significant toxicity at therapeuticdoses, and displayed clinical responses in ovarian andcolorectal cancer (12).

Myc is a nuclear phosphoprotein in the basic/helix-loop-helix/leucine zipper family of transcription factors that

associates with Max to activate proliferative genes (13).Myc protein accelerates cells into S phase by inductionof cyclin D1 (14), which increases shortly after estrogeninduction of MYC mRNA expression in mammary cells(15) via an atypical estrogen response element (16).

As a corollary of MYC overexpression in breast cancers(4), MYC antisense oligonucleotides inhibited estrogen-stimulated MCF7 breast cancer cell proliferation, as wellas the estrogen-independent MDA-MB-231 breast cancerline (17). Correspondingly, induction of MYC expressionin antiestrogen-arrested cells mimicked the effects ofestrogen by reinitiating cell cycle progression (18), whileconstitutive overexpression of Myc protein conferredresistance to antiestrogen treatment in human MCF7breast cancer cells (19). In the cell signaling pathway,antisense inhibition of MYC mRNA in MCF7 breastcancer cells inhibited cyclin D1 expression and cellproliferation (15, 20), even though MYC gene inductiondid not increase cyclin D1 expression. Thus, while cyclinD1 expression is dependent on Myc protein, MYC geneexpression is not limited by cyclin D1.

Clinical examination and mammography, the currentlyaccepted breast cancer screening methods, miss almosthalf of breast cancers in women younger than 40 years,approximately one-quarter of cancers in women aged 40-49 years, and one-fifth of cancers in women over 50 yearsold (21). The breast is the most common site of noncu-taneous cancer in US women (22). Moreover, if an abnor-mality is found, an invasive diagnostic procedure muststill be performed to determine if the breast containsatypia or cancer. Despite recent improvements in BI-RADS standardization of mammography (including theuse of two views of the breast rather than one, spot com-pression, and digital images) that have improved sensi-

* Corresponding author: Dr. Eric Wickstrom, Department ofBiochemistry and Molecular Pharmacology, Thomas JeffersonUniversity, 219 Bluemle Life Sciences Building, 233 S. 10th St.,Philadelphia PA 19107-5541, voice: 215-955-4578, fax: 215-955-4580 or 215-923-9214, [email protected], website: http://tesla.jci.tju.edu.

† Department of Biochemistry and Molecular Pharmacology,Thomas Jefferson University.

‡ Department of Radiology, Thomas Jefferson University.§ Kimmel Cancer Center, Thomas Jefferson University.| University of Missouri.

70 Bioconjugate Chem. 2005, 16, 70−79

10.1021/bc0497923 CCC: $30.25 © 2005 American Chemical SocietyPublished on Web 12/31/2004

tivity and specificity (23), 66-85% of women with suspi-cious mammograms who undergo surgery are found tohave a benign abnormality (24). In many other patientsrecently determined to have a benign mammogram,clinical examination reveals a mass found to be malig-nant.

A noninvasive method to evaluate women for thepresence of atypia and/or cancer of the breast would bevery beneficial. SestaMIBI has demonstrated utility inimaging lesions in a dense breast, but is limited by thefact that other cell types with high mitochondrial activityavidly take up the tracer, leading to false positive resultsin breasts with inflammation/infection (25), false nega-tives due to sensitivity limits of the technology, in whichtumors <8 mm in diameter are difficult to visualize (26),and the potential for false negatives with well differenti-ated, slowly growing tumors due to their lower mitochon-drial content.

Recent evidence from gene expression profiling sug-gests that alterations in mRNA conferring the potentialfor invasive breast cancer are already present in prein-vasive disease, supporting the rationale for this approach(27). However, transcription profiles of breast cancertissue by microarray analysis have not yet identifieddetectable overexpression of those markers (28), and thatapproach requires an invasive biopsy. As a step incharacterizing preinvasive disease, such as ductal car-cinoma in situ, it might be helpful to identify sites of MYCoverexpression externally, without a biopsy.

Therefore, we hypothesized that noninvasive imagingof a radiolabeled oligonucleotide hybridized to MYCmRNA inside breast cancer cells might identify sites ofneoplastic transformation. Previously, a [99mTc]6-hydrazi-nonicotinate-conjugated MYC antisense oligonucleotidephosphorothioate failed to show differences in uptakeamong cell lines with high, normal, or low levels of MYCmRNA (29). Those negative results might have been dueto nonspecific affinity of phosphorothioate oligonucle-otides for proteins, limiting efflux of unbound label, andto destruction of the oncogene message being measuredby RNase H attack on the RNA target due to hybridiza-tion of the labeled antisense DNA, or to lack of a receptortargeting ligand. While specific tumor uptake of [111In]-MYC phosphorothioate (30) and [68Ga]KRAS phospho-rothioate (31) have been asserted, those reports have notbeen replicated as yet.

Morpholino phosphorodiamidates provide a usefulalternative to phosphorothioates as hybridization agents,but suffer from poor uptake (32). However, radiolabeledmorpholino oligonucleotides have exhibited good phar-macokinetic and tissue distribution properties (33). Avoid-ance of cytosines in morpholino sequences reduces renal

accumulation (34), while conjugation of basic peptideselevates cellular uptake of morpholino oligomers (35).

To address those drawbacks, we turned to peptidenucleic acids (PNAs), which hybridize more tightly andmore specifically to RNA than morpholino oligonucle-otides (36, 37), resist nuclease attack, and demonstrateantisense activity upon microinjection of PNAs intocellular nuclei (38). RNA hybridized to uncharged oligo-nucleotide derivatives, such as PNA, is not recognizedby RNase H. Therefore, PNA does not catalyze degrada-tion of its analyte, the bound RNA (39), but inhibitsmRNA translation solely by hybridization arrest, andthus provides an opportunity for diagnostic application(38). The requirement for intracellular PNA microinjec-tion, however, stems from their poor cellular uptake,which was reported to be 10 times less efficient thanuptake of phosphorothioates in a variety of mammaliancells (40).

Cellular uptake of PNAs can be improved by theaddition of ligands (41). Previously we observed thatsynthesis of an IGF1R PNA dodecamer with an N-terminal D-peptide analogue of IGF1, D(CysSerLysCys),provided cell-type specificity, and increased cellularuptake 5-10-fold in BALB/c3T3 cells transformed withhuman IGF1R (42) and in human IGF1R-overexpressingMCF7 breast cancer cells (43). Only background levelsof uptake were seen with control cell lines or controlpeptides. In prostate cancer cells, for comparison, addi-tion of dihydrotestosterone or a nuclear localizationpeptide to a MYC antisense PNA permitted some nuclearlocalization and Myc reduction in LNCaP cells expressingandrogen receptor after 24 h exposure to 10 µM PNA (44).It appears that the use of a peptide analogue specific fora cell surface receptor is more effective than a steroidcapable of binding to a cytoplasmic protein after unas-sisted uptake.

We have pursued this cell-specific approach to enableapplication of PNAs as gene expression diagnostics invivo, developing methods for the synthesis of peptide-PNA-peptide chimeras, which exhibit the same meltingtemperatures with complementary RNA targets as doPNAs alone without peptides (45). For noninvasivescintigraphic imaging of oncogene mRNA in malignantlesions, we developed a method to label PNA-peptidehybridization probes with 99mTc by including a chelatingpeptide GlyD(Ala)GlyGlyAba (Aba ) γ-aminobutanoate)(TP3654) (46) coupled by continuous solid-phase synthe-sis to the N-terminus of PNA-AEEA-D(CysSerLysCys)(AEEA ) aminoethoxyethoxyaceate) (45). Fluoresceinyl-GlyD(Ala)GlyGlyAba-CCND1 PNA-AEEA-D(CysSerLy-sCys) lowered cyclin D1 protein in IGF1R-overexpressingMCF7 xenografts in immunocompromised mice upon

Figure 1. [99mTc]AcGlyD(Ala)GlyGlyAba-MYC PNA-AEEA-D(CysSerLysCys), WT4219, designed to bind to the receptor for IGF1,internalize, and hybridize with MYC mRNA. Scintigraphic imaging of γ-particles emitted upon decay of 99mTc might identify sitesof high MYC mRNA expression.

Noninvasive MYC mRNA Imaging Bioconjugate Chem., Vol. 16, No. 1, 2005 71

intratumoral injection (43). The γ-emitting [99mTc]Ac-GlyD(Ala)GlyGlyAba-CCND1 PNA-AEEA-D(CysSer-LysCys) visualized IGF1R-overexpressing, CCND1-over-expressing MCF7 xenografts in immunocompromisedmice at 12 and 24 h after intravenous administration.In all of those experiments, control PNA and peptidesequences were unable to produce similar images (43).

Because MYC mRNA is overexpressed in a broadspectrum of cancers, it might be a valuable target fordiagnostic imaging in many cases. We report here imag-ing of MYC mRNA levels in human estrogen receptor-positive MCF7:IGF1R breast cancer xenografts in im-munocompromised mice following intravenous admin-istration of [99mTc]AcGlyD(Ala)GlyGlyAba-MYC PNA-AEEA-D(CysSerLysCys) chimera, designed to bind to thereceptor for IGF1, internalize, and hybridize with MYCmRNA (Figure 1).

MATERIALS AND METHODS

Peptide-PNA-Peptide Synthesis. The peptide-PNA-peptide chimeras were assembled (Scheme 1),purified, and characterized as described previously (45).Briefly, the IGF1 analogue D(CysSerLysCys), or themismatch control D(CysAlaAlaCys), was assembled byFmoc coupling on NovaSyn TGR resin (0.2-0.3 mmol/g)(Novabiochem, San Diego CA) on an Applied Biosystems430A peptide synthesizer. Then the linker Fmoc-AEEAand PNA monomers (Applied Biosystems, Foster CityCA) were sequentially coupled to the N-terminus of thepeptide-resin on an Applied Biosystems 8909 DNAsynthesizer, running the manufacturer’s PNA 2 µmolprotocol. Finally, the chelator peptide Fmoc-GlyD(Ala)-GlyGlyAba (Novabiochem) was coupled to the N-terminusof the PNA-peptide using a long coupling cycle protocol(45), then acetylated. The cysteine residues were cyclizedon solid phase with 10 equivalents of I2 in Me2NCHO for4 h at room temperature (45).

The chimeras were cleaved and deprotected with 85%CF3CO2H, 5% CH2Cl2, 9.5% m-cresol, and 0.5% Et3SiHfor 2 h at room temperature (45), then purified byreversed phase liquid chromatography on a 10 × 250 mmAlltima C18 column eluted with a gradient linear 25 min.from 5% to 45% CH3CN in aqueous 0.1% CF3CO2H, at 1mL/min, at 50 °C, monitored at 260 nm. Matrix-assistedlaser desorption ionization time-of-flight mass spectrom-etry (MALDI-TOF MS, Ciphergen Corp., Fremont, CA)was carried out from an R-cyano-hydroxycinnamic acidmatrix excited at 338 nm, and calibrated with porcineneuropeptide Y.

Cell Lines and Xenografts. Human MCF7:IGF1Restrogen receptor-positive breast cancer cells, clone 17,transformed to express 1 × 106 IGF1R/cell constitutivelyfrom a cytomegalovirus promoter (47) were maintainedin DMEM plus 5% calf serum, 50 U/mL penicillin, 5 µg/mL streptomycin, 2 mM glutamine, and 7.5 nM 17-â-estradiol (Sigma) at 37 °C under 5% CO2. For tumorinduction, 5-6 × 106 cells in 0.2 mL of culture mediumwere implanted intramuscularly through a sterile 27gauge needle into the thighs of female Balb/c nude miceobtained from NIH. Tumors were allowed to grow to nomore than 0.5 cm in diameter. Each injection included10 mg of Matrigel. A 60-day release pellet containing 4.5mg of 17-â-estradiol (Innovative Research of America,Sarasota, FL) was implanted subdermally in each mouse.All animal studies were conducted in accordance withfederal and state guidelines governing the laboratory useof animals, and under approved protocols reviewed bythe Animal Care and Use Committee at Thomas Jeffer-son University. All animals were anesthetized by ap-proved methods, and when required, the animals wererestrained using methods and devices specifically de-signed to provide a minimum of discomfort to the animal.Animals were euthanized in a halothane chamber, con-

Scheme 1. Assembly of Chelator Peptide-MYC PNA-Peptides

72 Bioconjugate Chem., Vol. 16, No. 1, 2005 Tian et al.

sistent with USDA regulations and American VeterinaryMedical Association recommendations.

Radiolabeling. Purified peptide-PNA-peptide chi-meras were labeled with 99mTc (Scheme 2) essentially asdescribed previously (48). Briefly, 20 µg of peptide-PNA-peptide chimera and 45-60 µg of SnCl2‚2H2O werevortexed in 20 µL of 0.05 M HCl. Next, 12 mCi ofNa99mTcO4 freshly eluted from the 99mTc generator with200 µL of 0.15 M NaCl were vortexed with 600 µL of 0.05M Na3PO4, pH 12, 0.1% Tween 80. The radionuclidemixture was then added to the peptide-PNA-peptide/SnCl2 solution and incubated for 30 min at room tem-perature. Finally, the solution was neutralized by theaddition of 1 mL of 0.05 M NaH2PO4, pH 4.5.

Reaction mixtures were analyzed to determine free99mTc and chelated 99mTc by reversed phase liquid chro-matography on a 4.5 × 250 mm reversed phase C18microbond HPLC column (Rainin, Emeryville, CA) coupledto a UV detector, NaI (Tl) radioactivity monitor, and arate meter. The column was eluted with a 28 min.gradient from 10% to 100% CH3CN in aqueous 0.1% CF3-CO2H, at 1 mL/min, at 25 °C. Unchelated free 99mTc (Rf1.0) was also determined by instant thin-layer chroma-tography on silica gel (ITLC-SG, Gelman Sciences, AnnArbor, MI) developed with methylethyl ketone. Colloidformation (Rf 0.0) was determined on ITLC-SG devel-oped with pyridine/HOAc/H2O (3:5:1.5). These prepara-tions were stable at 22 °C for more than 4 h, asdetermined by HPLC, and were stable to challenges with100-fold molar excesses of diethylenetriaminepentaaceticacid, human serum albumin, or cysteine.

Systemic Administration and Tissue Distributionof Labeled Probes. To assess chimera distribution andimaging, 7.4-11.1 MBq (0.2-0.3 mCi) of the [99mTc]-peptide-PNA-peptides in 0.2 mL of sterile 0.1 M Na2-HPO4, pH 7, was administered to groups of five mice eachthrough a lateral tail vein using a sterile 27 gauge needle.For the 24 h distribution, 29.6-34.2 MBq (0.8-0.9 mCi)of probe was administered. At 4 and 24 h postinjection,mice were lightly anesthetized, then imaged using aStarcam (GE Medical, Milwaukee, WI) gamma cameraequipped with a parallel hole collimator. For each image,300 000 counts were collected. Digital scanning of region-of-interest intensities with the interfaced Entegra com-puter (GE Medical) across each scintigraphic image fromthe tumor-free left flank to the tumor-bearing right flankprovided quantitation of tumor images.

Mice were then euthanized, and tissues were dissected.These were washed free of blood, blotted dry, and

weighed, and radioactivity associated with each tissuewas counted in an automatic gamma counter (PackardSeries 5000, Meridien, CT), together with a standardradioactive solution of a known quantity prepared at thetime of injection. Results were expressed as percent ofinjected dose per gram of tissue (% I.D/g).

In Vivo Stability. Urine was collected up to 2-3 hpostinjection from groups of 5 mice injected with [99mTc]-peptide-PNA-peptide probes for assessment of probestability. The urine samples were combined, sedimentedat 2000g for 10 min, filtered through Millipore HAWP025 membranes with 0.22 µm pores, and analyzed by RP-HPLC as described in Radiolabeling above.

Statistical Methods. Statistical analysis of differ-ences among groups was determined by applying thestandard error of the mean, Student’s t test, Kruskal andWallis’s one-way analysis of variance on ranks, or theHolm-Sidak pairwise multiple comparison procedure,using SigmaStat 3.0 (SPSS, Chicago, IL).

RESULTSPeptide-PNA-Peptides. We prepared a cyclized

peptide-PNA-peptide chimera AcGlyD(Ala)GlyGlyAba-GCATCGTCGCGG-AEEA-D(CysSerLysCys), WT4219, spe-cific for MYC mRNA and the IGF1 receptor (Figure 1).Preparative RP-HPLC of the crude product yielded amain peak containing 98.05% of the absorbance at 260nm (Figure 2A). The overall yield following preparativeRP-HPLC was 27.2%. MALDI-TOF MS confirmed theidentity of the main product peak at 4221.6 Da (Figure2B). Similarly, we prepared a MYC PNA 4-mismatchcontrol, AcGlyD(Ala)GlyGlyAba-GCATGTCTGCGG-AEEA-D(CysSerLysCys), WT4235, with 30.6% overall yieldfollowing preparative RP-HPLC (Figure 3A), and MALDI-TOF mass of 4234.7 Da (Figure 3B).

[99mTc]Peptide-PNA-Peptides. Twenty microgramaliquots of the MYC peptide-PNA-peptide antisenseprobe, WT4219, labeled well at 22 °C (Figure 4A). Free99mTc was only 0.36%, and colloids were only 4.84%. ThePNA mismatch, WT4235, labeled well also (Figure 4B).Free 99mTc was 0.99%, and colloids were 4.84%.

Systemic Administration. To test our hypothesisthat a complementary [99mTc]peptide-PNA-peptide probespecific for MYC mRNA and the IGF1 receptor couldvisualize a MYC-expressing tumor, we administeredapproximately 0.5 mCi [99mTc]peptide PNA-peptide MYCprobes to cohorts of five nude mice bearing approximately0.5 cm MCF7:IGF1R xenografts to determine the speci-ficity and sensitivity of scintigraphic imaging 4 h and 24

Scheme 2 99mTc Labeling of Peptide-PNA-peptides


h after administration (Figure 5). We previously testedwhether the PNA-free chelator plus IGF1 analoguecontrol probe would bind to tumor cells expressing highlevels of IGF1 receptor, which it did not (43).

To test whether a probe containing four central PNAmismatches would bind to tumor cells expressing highlevels of IGF1 receptor and MYC mRNA, we adminis-tered the PNA mismatch control, WT4235. We did notobserve tumor signals at 4 h or 24 h postinjection.Finally, to test whether the complementary probe withcorrect IGF1 analogue would bind to tumor cells express-ing high levels of IGF1 receptor and MYC mRNA, weadministered the PNA antisense chimera, WT4219. Inthis case, we observed faint tumor signals at 4 h andintermediate tumor signals at 24 h postinjection.

Digital scanning of the tumor site region-of-interest onthe right flank vs the mirror image tumor-free region-of-interest on the left flank enabled quantitation of tumorimages. The ratios of tumor site:control site intensitiesare plotted in Figure 6 as a function of time. One pre-sumes that any ratio greater than 1 implies some prefer-

ential probe concentration. No tumor image was obviousin Figure 5 following administration of the PNA mis-match control, WT4235, which includes the IGF1 peptideanalogue, but the digitized tumor site:control site ratiowas 3.33 ( 0.30 at 4 h, and 2.72 ( 0.44 at 24 h. A fainttumor image was apparent in Figure 5 four h after admin-istration of the antisense probe, WT4219, which corre-lated with the tumor site:control site ratio of 4.35 ( 0.58.At 24 h postinjection, the ratio increased to 7.01 ( 0.90.

Kruskal-Wallis one-way analysis of variance on ranksdetermined that the four groups were significantly dif-ferent from each other (p < 0.001). When all four groupswere compared by the Holm-Sidak method, the anti-sense group was not significantly different from themismatch group at 4 h postinjection (p ) 0.244). How-ever, at 24 h postinjection, Holm-Sidak comparisonrevealed a statistically significant difference (p <0.001)between the antisense group and the mismatch group.The test performed had a power of 0.985 with R ) 0.050.

In Vivo Stability. RP-HPLC of radioactivity in thecombined 3-h urine sample from mice injected with [99m-

Figure 2. Characterization of cyclized MYC antisense chimera AcGlyD(Ala)GlyGlyAba-GCATCGTCGCGG-AEEA-D(CysSerLysCys),WT4219. A. Preparative reversed phase liquid chromatography of cyclized chimera on a 10 × 250 mm Alltima C18 column elutedwith a linear 30 min. gradient from 5% to 45% CH3CN in aqueous 0.1% CF3CO2H, at 1 mL/min, at 50 °C, monitored at 260 nm. B.MALDI-TOF mass spectrum of chimera in a matrix of R-cyano-hydroxycinnamic acid. Experimental mass: 4221.6 Da; calculatedmass: 4219.0 Da.

Figure 3. Characterization of cyclized MYC mismatch chimera AcGlyD(Ala)GlyGlyAba-GCATGTCTGCGG-AEEA-D(CysSerLysCys),WT4235. A. Preparative C18 HPLC of cyclized chimera as in Figure 2. B. MALDI-TOF mass spectrum of chimera as in Figure 2.Experimental mass: 4234.7 Da; calculated mass: 4235.0 Da.


Tc]MYC antisense probe, WT4219, showed no void vol-ume peak of free 99mTc, but only an intact probe peak at10.7 min (Figure 7, left). RP-HPLC of radioactivity in thecombined 2-h urine sample from mice injected with[99mTc]MYC mismatch probe revealed a void volume peakof free 99mTc, 44% of radioactivity, and a probe peak at10.3 min with 56% of the radioactivity (Figure 7, right).Breakdown fragments of the peptide-PNA-peptide probewere not detected from either probe.

Tissue Distribution. Tissue distribution of eachprobe was measured in all subjects at 4 and 24 h afteradministration. Table 1 presents the data for the anti-sense group and the mismatch group at 4 h, while Table

2 shows the 24 h results. As the time after injectionelapsed, the radioactivity in all tissues decreased, includ-ing that in the tumors. However, the tumor to blood andtumor-to-muscle ratios increased for WT4219 as timewent on. This is consistent with the scintigraphic imagesin Figure 5 and the tumor (right)-to-muscle (left) imageintensity ratios provided in Figure 6, which were higherthan the tumor/muscle distribution ratios from scintil-lation counting for WT4219. While the tumors werereadily detectable by scintigraphy with WT4219, but notwith WT4235, the tumor distribution counts of % injecteddose/g for the antisense group and the mismatch groupwere not remarkably different, except for the heart. The

Figure 4. Analysis of [99mTc]peptide-PNA-peptides. An aliquot of each labeling reaction was analyzed by RP-HPLC on a 10 × 250mm Rainin Microbond C18 column eluted with a 28 min gradient from 10% to 100% CH3CN in aqueous 0.1% CF3CO2H, at 1 mL/min,at 25 °C. %B (CH3CN) is shown on the right y-axis, and NaI (Tl) radiometric gamma emission is shown on the left y-axis. Left:antisense probe, WT4219. The single labeled peak eluted at 10.37 min. Right: mismatch probe, WT4235. The single labeled peakeluted at 10.25 min.

Figure 5. Scintigraphic images of γ-particles emitted by decaying 99mTc in immunocompromised mice carrying human MCF7:IGF1R ER+ breast tumor cell xenografts at 4 h and 24 h after injection of [99mTc]peptide-PNA-peptides. Left: [99mTc]MYC PNAantisense probe, WT4219; right: [99mTc]MYC PNA mismatch control, WT4235.


increased accumulation of the mismatch probe WT4235in heart tissue invited a BLAST search with the WT4235sequence for heart-specific or mitochondrial-specific mu-rine mRNAs. Unfortunately, no complementary homolo-gies were identified, so the concentration of mismatchprobe in the heart remains unexplained.

The tissue distribution data indicated that renal excre-tion was the primary route of elimination and renaluptake was highest among all tissues, but the extentdiffered from agent to agent.

DISCUSSION

In this report we tested the feasibility of using a [99m-Tc]chelator peptide-MYC PNA receptor-targeting hybrid-ization probe to image MYC mRNA at modest copynumber in ER+ breast cancer cells that overexpressIGF1R, a receptor that is overexpressed is a highpercentage of breast cancer cells.

Quantitative reverse transcription-polymerase chainreaction measurements of MYC mRNA in total MCF7:IGF1R cell RNA revealed 4-fold inhibition after prean-nealing with 0.1 µM N-GlyD(Ala)GlyGlyAba-MYC PNA-Gly4D(CysSerLysCys) probe before adding PCR primers,and 8-fold inhibition after preannealing with 1.0 µMprobe (39). The MYC mismatch control had no effect,consistent with the hypothesis that the complementaryPNA probe would bind strongly to the mRNA target. Thatprobe was essentially the same as WT4219, except forsubstitution of Gly4 for the AEEA linker (39). Thespecificity of RT inhibition implies that the peptide-PNA-peptides hybridize to their intended MYC mRNAtarget.

Previously, denaturing NaDodSO4 gel electrophoresis(43) of a comparable [99mTc]peptide-CCND1 PNA-pep-tide probe showed a radioactive band that co-electro-phoresed with the main radioactive band from the[99mTc]CCND1 probe labeling mixture. Similarly, fluo-rescence microscopy illustrated that the peptide-CCND1PNA-peptide probes were taken up inside MCF7:IGF1Rbreast cancer cells, if and only if the IGF1 peptide

analogue sequence was correct (43), as was reportedpreviously for p6 cells (42).

We carried out a variety of tests of the hypothesis thatoverexpression of MYC mRNA in MCF7:IGF1R breastcancer xenografts could be detected specifically andnoninvasively by scintigraphic imaging of 99mTc-peptide-PNA-peptide probes. We previously measured MYConcogene mRNA copy number in MCF7:IGF1R humanbreast cancer cells by quantitative reverse transcription-polymerase chain reaction (43). Comparison of thresholdcycle numbers for MYC mRNA with those for TATA-boxbinding protein mRNA yielded an average ratio of 8 forMCF7:IGF1R cells over two logs of RNA template. Onthe basis of the previously reported TATA-box bindingprotein mRNA copies per breast cancer cell of 1000-2000(49), we estimated approximately 8000 MYC mRNAs perMCF7:IGF1R cell. Assuming 8000 mRNAs per cell and109 cells per gram of tumor, there would be an estimated8 × 1012 mRNA copies available for binding in a 1 gtumor. Our data suggest that 0.1% of the injected [99mTc]-WT4219 antisense probe (specific activity 2 Ci/µmol) wasassociated with the tumors at 24 h. This suggests thatapproximately 3 × 1011 molecules of [99mTc]WT4219 werebound to the mRNA in a 1 g tumor, or about 4% of theMYC mRNAs, despite nonuniform distribution of radio-activity in the tumor. In a high specific activity or carrier-free preparation, this would be equivalent to 500 µCi of99mTc, suggesting the possibility of up to 500 times greatersensitivity than we observed in this initial study.

To estimate the fraction of internalized PNA probesthat could be hybridized to available MYC mRNAs in livetumor cells, one must begin with the observed Tm ofapproximately 80 °C for 2.5 µM peptide-PNA-peptide12-mers with 2.5 µM RNA (45). A copy number ofapproximately 8000 MYC mRNAs per 1 pL cell translatesto 13 nM, while the [99mTc]WT4219 appeared to be lessthan 1/10 of that, 1 nM. The Tm, the probe concentration,and the 13-fold excess mRNA concentration enableprediction (36) of virtually complete hybridization ofavailable intracellular probe.

Interestingly, concentration of label in tumors was veryfaint at 4 h after injection of the antisense PNA MYCsequence with IGF1 analogue, WT4219, although thedigital scan revealed detectable concentrations of label.We interpret that observation to mean that uptake,intracellular trafficking, hybridization of label, and effluxof unbound label are all slow processes that do not reveala strong tumor signal at 4 h. Because labeled antisensePNA in the tumors could be visualized at 24 h, wehypothesize that 24 h was sufficient for unbound probeto efflux from cells and to be excreted. With respect tothe hybridized label, bound PNA probe dissociates neg-ligibly from complementary RNA by 12-24 h (25, 36).In the end, we found that the MCF7:IGF1R xenograftsthat overexpress MYC mRNA could be imaged reliablyand specifically at 24 h postinjection.

The [99mTc]MYC mismatch probe was also adminis-tered to immunocompromised mice to assess metabolismin vivo. Urinalysis by RP-HPLC (Figure 7), showingintact [99mTc]MYC antisense and mismatch probes, im-plied reasonable stability of the peptide-PNA agent invivo, consistent with the hypothesis that a [99mTc]D-peptide-PNA would be resistant to proteases and nu-cleases in blood and tissues. In the case of the mismatchprobe, free 99mTc void volume peak probably resulted fromreoxidation of probe-bound Tc(III) to free Tc(VII) in theliver. The lack of a free 99mTc void volume peak in thecase of the antisense probe is remarkable. One mayspeculate that the antisense probe was only excreted

Figure 6. Tumor site:control site gamma intensity ratios aftersystemic administration of [99mTc]peptide-PNA-peptide chi-meras for all subjects in Figure 5. red, [99mTc]MYC PNAmismatch control, WT4235; green, [99mTc]MYC PNA antisenseprobe, WT4219.


upon the first pass through the kidney, and that anti-sense probes that distributed to tissues did not re-enterthe bloodstream to be metabolized by the liver, at leastup to 3 h.

The tissue distribution and urinalysis results werecomparable to our previous observations with [99mTc]-GlyD(Ala)GlyGlyAba-GCATCGTCGCGG, WT4219 lack-ing the IGF1 analogue (39). The tissue distributionresults were also comparable with those for [99mTc]-AcGlyD(Ala)GlyGlyAba-CCND1 PNA-IGF1 analoguesand controls (43). As noted above, a BLAST search withthe mismatch sequence did not uncover any complemen-tary cardiac or mitochondrial mRNAs.

Although there was no significant difference in tumor:muscle ratios between the MYC probe and its mismatchcontrol, the MCF7:IGF1R xenografts were imaged clearly

with WT4219, but not with WT4235. That is because thetissue distribution data report radioactivity in the entireexcised masses, which include some surrounding normaltissue, the actively proliferating cells on the peripheryof the tumors, and the necrotic cores. The radioactiveMYC probes were predicted to concentrate in the activelyproliferating cells. However, the surrounding normaltissues, which do not express human MYC mRNA, andthe nectrotic cores, which exhibit poor uptake of macro-molecules (50) and are not proliferating, were not dis-sected away before measuring tissue radioactivity. As aresult, the apparent % uptake of 99mTc/g tumor wasdiluted by inclusion of extra nonproliferating tissue.Hence, the tumor/muscle ratios of MYC probe radioactiv-ity were reduced to the range of the mismatch probe. Thissame difference between clear scintigraphic images andmodest % uptake of 99mTc/g tumor was also observedearlier for [99mTc]GlyD(Ala)GlyGlyAba-vasoactive intes-tinal peptide, which revealed occult tumors in patientsdespite apparent tumor uptake of only 0.3% injecteddose/g in xenografts in immunocompromised mice (46,48, 51), and for [99mTc]AcGlyD(Ala)GlyGlyAba-CCND1PNA-IGF1 analogues and controls in MCF7:IGF1R xe-nografts (43). Digital scanning, however, of scintigraphicintensities determined the radioactivity per pixel in thetumor region-of-interest, revealing MYC mRNA expres-sion in the actively proliferating cells on the peripheryof the tumors. In this case, the necrotic centers presum-ably did not contribute to the measurement, eliminatingthe possibility of label dilution. Thus, the tumor/contra-lateral scintifigraphic intensity ratios in Figure 6 prob-ably provided more accurate results than the scintillationcounting tissue distribution ratios in Tables 1 and 2.

High grade renal cell carcinomas exhibit elevatedcytoplasmic Myc protein, but not in the nuclei, and notin normal renal cells (40, 52). Therefore, concentrationof the PNA antisense probe WT4219 in the kidneys wasprobably not due to labeling of MYC mRNA in kidneycells, but to nonspecific binding to negative charges onrenal parenchyma. Simultaneous L-lysine administrationhas been shown to reduce renal accumulation of [111In]-DTPA-octreotide in rats (53). Preliminary results from astudy underway in our laboratory with our [99mTc]CCND1probe administered to tumor-bearing mice are consistentwith that report.

Our observation of scintigraphic imaging of MYCmRNA expression in xenografts could be applicable to

Figure 7. Analytical HPLC of [99mTc]peptide-PNA-peptide probes that were recovered from mouse urine 2-3 h after administration,chromatographed as in Figure 4 above. Left, [99mTc]MYC antisense probe, WT4219, 3 h after administration; right, [99mTc]MYCmismatch probe, WT4235, 2 h after administration.

Table 1. Tissue Distribution (% injected dose/g) of the[99mTc]MYC Antisense Chimera, WT4219, and the[99mTc]MYC Mismatch Chimera, WT4235, at 4 h afterSystemic Administration (n ) 5)

tissues antisense mismatch null prob.

muscle 0.08 ( 0.02 0.11 ( 0.03 0.11intestine 0.10 ( 0.03 0.11 ( 0.01 0.18heart 0.09 ( 0.02 0.68 ( 0.15 0.00003lungs 0.52 ( 0.19 0.70 ( 0.27 0.25blood 0.18 ( 0.03 0.24 ( 0.03 0.01spleen 3.64 ( 1.71 4.44 ( 0.98 0.39kidneys 31.54 ( 3.14 37.47 ( 3.42 0.02liver 5.55 ( 0.77 3.43 ( 0.51 0.00091tumor 0.20 ( 0.05 0.23 ( 0.05 0.47T/M ratio 2.57 ( 0.96 2.26 ( 0.78 0.59T/B ratio 1.14 ( 0.17 0.97 ( 0.29 0.30

Table 2. Tissue Distribution (% injected dose/g) of the[99mTc]MYC Antisense chimera, WT4219, and the[99mTc]MYC Mismatch Chimera, WT4235, at 24 h afterSystemic Administration (n ) 5)

tissues antisense mismatch null prob.

muscle 0.04 ( 0.01 0.05 ( 0.01 0.45intestine 0.04 ( 0.00 0.05 ( 0.00 0.008heart 0.03 ( 0.00 0.24 ( 0.03 0.0000004lungs 0.15 ( 0.04 0.25 ( 0.03 0.002blood 0.04 ( 0.01 0.05 ( 0.01 0.06spleen 3.47 ( 0.88 3.32 ( 1.41 0.84kidneys 21.65 ( 6.50 20.06 ( 4.64 0.67liver 4.95 ( 0.58 2.93 ( 0.75 0.001tumor 0.10 ( 0.03 0.11 ( 0.03 0.63T/M ratio 2.59 ( 0.88 2.19 ( 0.30 0.37T/B ratio 2.48 ( 0.56 2.09 ( 0.55 0.30


patients at risk for new or recurrent breast cancer,including those with a strong family history of breastcancer, or when standard detection techniques provideambiguous results. Imaging of mRNA expression withchelator-PNA-peptide chimeras labeled with 99mT, 64Cu,or other radionuclides might also be applicable to otheroncogenes or to other cell surface receptors.

Targeting altered expression of oncogenes provides thepromise of specificity, ultimately leading to the elimina-tion of diagnostic surgical biopsy. MYC overexpressionis a common event in and highly associated with thepresence of breast cancer. While not sufficiently sensitiveto be used alone, noninvasive measurement of mRNAexpression by one or more oncogenes to confirm thepresence or absence of cancer by γ-particle emission orby positron emission tomography when an imaging studyis abnormal provides a mechanism for more accuratediagnosis of breast cancer.

ACKNOWLEDGMENT

We thank Dr. Eva Surmacz for her advice and the giftof MCF7 cells that constitutively overexpress IGF1R,Zuping Qu for maintaining the MCF7 cells under avariety of conditions, Dr. Zhi-xian Lu for assembly oflinear peptide-resin supports, and Dr. Richard Wassellfor assistance in measuring mass spectra of peptide-PNA-peptides. This work was supported by DOEER63055 to E.W. and NIH HL59769 to M.L.T.

LITERATURE CITED

(1) Duesberg, P. H., Bister, K., and Vogt, P. K. (1977) The RNAof avian acute leukemia virus MC29. Proc. Natl. Acad. Sci.U. S. A. 74, 4320-4324.

(2) Heikkila, R., Schwab, G., Wickstrom, E., Loke, S. L.,Pluznik, D. H., Watt, R., and Neckers, L. M. (1987) A c-mycantisense oligodeoxynucleotide inhibits entry into S phase butnot progress from G0 to G1. Nature 328, 445-449.

(3) Wickstrom, E. L., Bacon, T. A., Gonzalez, A., Freeman, D.L., Lyman, G. H., and Wickstrom, E. (1988) Human promy-elocytic leukemia HL-60 cell proliferation and c-myc proteinexpression are inhibited by an antisense pentadecadeoxy-nucleotide targeted against c-myc mRNA. Proc. Natl. Acad.Sci. U. S. A. 85, 1028-1032.

(4) Orr, M. S., Fornari, F. A., Randolph, J. K., and Gewirtz, D.A. (1995) Transcriptional down-regulation of c-myc expressionin the MCF-7 breast tumor cell line by the topoisomerase IIinhibitor, VM-26. Biochim. Biophys Acta 1262, 139-145.

(5) Schmitz-Drager, B. J., Schulz, W. A., Jurgens, B., Gerharz,C. D., van Roeyen, C. R., Bultel, H., Ebert, T., and Acker-mann, R. (1997) c-myc in bladder cancer. Clinical findingsand analysis of mechanism. Urol. Res. 25 Suppl 1, S45-S49.

(6) Mizutani, Y., Bonavida, B., Fukumoto, M., and Yoshida, O.(1995) Enhanced susceptibility of c-myc antisense oligonucle-otide-treated human renal cell carcinoma cells to lysis byperipheral blood lymphocytes. J. Immunother. EmphasisTumor Immunol. 17, 78-87.

(7) Rygaard, K., Vindelov, L. L., and Spang-Thomsen, M. (1993)Expression of myc family oncoproteins in small-cell lung-cancer cell lines and xenografts. Int. J. Cancer 54, 144-152.

(8) Hernandez, L., Hernandez, S., Bea, S., Pinyol, M., Ferrer,A., Bosch, F., Nadal, A., Fernandez, P. L., Palacin, A.,Montserrat, E., and Campo, E. (1999) c-myc mRNA expres-sion and genomic alterations in mantle cell lymphomas andother nodal non-Hodgkin’s lymphomas. Leukemia 13, 2087-2093.

(9) Wickstrom, E., Bacon, T. A., and Wickstrom, E. L. (1992)Down-regulation of c-MYC antigen expression in lymphocytesof Eµ-c-myc transgenic mice treated with anti-c-myc DNAmethylphosphonates. Cancer Res. 52, 6741-6745.

(10) Huang, Y., Snyder, R., Kligshteyn, M., and Wickstrom, E.(1995) Prevention of tumor formation in a mouse model of

Burkitt’s lymphoma by 6 weeks of treatment with anti-c-mycDNA phosphorothioate. Molecular Medicine 1, 647-658.

(11) Smith, J. B., and Wickstrom, E. (1998) Antisense c-mycand immunostimulatory oligonucleotide inhibition of tum-origenesis in a murine B-cell lymphoma transplant model.J. Natl. Cancer Inst. 90, 1146-1154.

(12) Gelmon, K. A., Batist, G., Chi, K., Sandor, K., Webb, M.,D’Aloisio, S., Burge, C., Saltman, D., Goldie, J., and Miller,W. A dose escalation phase I study of c-MYC antisense incombination with cisplatin in the treatment of solid tumoursand lymphomas. Presented at the AACR-NCI-EORTC Inter-national Conference on Molecular Targets and Cancer Thera-peutics, Miami Beach, FL, Oct 29-Nov 2, 2001.

(13) Nair, S. K., and Burley, S. K. (2003) X-ray structures ofMyc-Max and Mad-Max recognizing DNA. Molecular basesof regulation by proto-oncogenic transcription factors. Cell112, 193-205.

(14) Perez-Roger, I., Kim, S. H., Griffiths, B., Sewing, A., andLand, H. (1999) Cyclins D1 and D2 mediate myc-inducedproliferation via sequestration of p27(Kip1) and p21(Cip1).Embo J. 18, 5310-5320.

(15) Doisneau-Sixou, S. F., Sergio, C. M., Carroll, J. S., Hui,R., Musgrove, E. A., and Sutherland, R. L. (2003) Estrogenand antiestrogen regulation of cell cycle progression in breastcancer cells. Endocr. Relat. Cancer 10, 179-186.

(16) Dubik, D., and Shiu, R. P. (1992) Mechanism of estrogenactivation of c-myc oncogene expression. Oncogene 7, 1587-1594.

(17) Watson, P. H., Pon, R. T., and Shiu, R. P. (1991) Inhibitionof c-myc expression by phosphorothioate antisense oligonucle-otide identifies a critical role for c-myc in the growth of humanbreast cancer. Cancer Res. 51, 3996-4000.

(18) Prall, O. W., Rogan, E. M., Musgrove, E. A., Watts, C. K.,and Sutherland, R. L. (1998) c-Myc or cyclin D1 mimicsestrogen effects on cyclin E-Cdk2 activation and cell cyclereentry. Mol. Cell Biol. 18, 4499-4508.

(19) Venditti, M., Iwasiow, B., Orr, F. W., and Shiu, R. P. (2002)C-myc gene expression alone is sufficient to confer resistanceto antiestrogen in human breast cancer cells. Int. J. Cancer99, 35-42.

(20) Carroll, J. S., Swarbrick, A., Musgrove, E. A., and Suth-erland, R. L. (2002) Mechanisms of growth arrest by c-mycantisense oligonucleotides in MCF-7 breast cancer cells:implications for the antiproliferative effects of antiestrogens.Cancer Res. 62, 3126-3131.

(21) Rosenberg, R. D., Hunt, W. C., Williamson, M. R., Gilliland,F. D., Wiest, P. W., Kelsey, C. A., Key, C. R., and Linver, M.N. (1998) Effects of age, breast density, ethnicity, andestrogen replacement therapy on screening mammographicsensitivity and cancer stage at diagnosis: review of 183, 134screening mammograms in Albuquerque, New Mexico. Ra-diology 209, 511-518.

(22) American Cancer Society (2004) Cancer Facts and Figures,American Cancer Society, Atlanta GA, http://www.cancer.org/downloads/STT/CAFF_finalPWSecured.pdf.

(23) Diekmann, F., Diekmann, S., Bollow, M., Hermann, K. G.,Richter, K., Heinlein, P., Schneider, W., and Hamm, B. (2004)Evaluation of a wavelet-based computer-assisted detectionsystem for identifying microcalcifications in digital full-fieldmammography. Acta Radiol. 45, 136-141.

(24) Fahy, B. N., Bold, R. J., Schneider, P. D., Khatri, V., andGoodnight, J. E., Jr. (2001) Cost-benefit analysis of biopsymethods for suspicious mammographic lesions; discussion994-5. Arch. Surg. 136, 990-994.

(25) Pappo, I., Horne, T., Weissberg, D., Wasserman, I., andOrda, R. (2000) The Usefulness of MIBI Scanning to DetectUnderlying Carcinoma in Women with Acute Mastitis. BreastJ. 6, 126-129.

(26) Lumachi, F., Zucchetta, P., Marzola, M. C., Ferretti, G.,Povolato, M., Paris, M. K., Brandes, A. A., and Bui, F. (2002)Positive predictive value of 99mTc sestamibi scintimammog-raphy in patients with nonpalpable, mammographicallydetected, suspicious, breast lesions. Nucl. Med. Commun. 23,1073-1078.

(27) Ma, X. J., Salunga, R., Tuggle, J. T., Gaudet, J., Enright,E., McQuary, P., Payette, T., Pistone, M., Stecker, K., Zhang,


B. M., Zhou, Y. X., Varnholt, H., Smith, B., Gadd, M.,Chatfield, E., Kessler, J., Baer, T. M., Erlander, M. G., andSgroi, D. C. (2003) Gene expression profiles of human breastcancer progression. Proc. Natl. Acad. Sci. U. S. A. 100, 5974-5979.

(28) Martin, K. J., Kritzman, B. M., Price, L. M., Koh, B., Kwan,C. P., Zhang, X., Mackay, A., O’Hare, M. J., Kaelin, C. M.,Mutter, G. L., Pardee, A. B., and Sager, R. (2000) Linkinggene expression patterns to therapeutic groups in breastcancer. Cancer Res. 60, 2232-2238.

(29) Stalteri, M. A., and Mather, S. J. (2000) In-vitro studieson 99m-Tc-labeled HYNIC-conjugated oligonucleotides. Nucl.Med. Commun. 21, 374.

(30) Dewanjee, M. K., Ghafouripour, A. K., Kapadvanjwala, M.,Dewanjee, S., Serafini, A. N., Lopez, D. M., and Sfakianakis,G. N. (1994) Noninvasive imaging of c-myc oncogene mes-senger RNA with indium-111-antisense probes in a mammarytumor-bearing mouse model. J. Nucl. Med. 35, 1054-1063.

(31) Roivainen, A., Tolvanen, T., Salomaki, S., Lendvai, G.,Velikyan, I., Numminen, P., Valila, M., Sipila, H., Bergstrom,M., Harkonen, P., Lonnberg, H., and Langstrom, B. (2004)68Ga-Labeled Oligonucleotides for In Vivo Imaging with PET.J. Nucl. Med. 45, 347-355.

(32) Summerton, J., and Weller, D. (1997) Morpholino antisenseoligomers: design, preparation, and properties. AntisenseNucleic Acid Drug Dev. 7, 187-195.

(33) Liu, G., Zhang, S., He, J., Liu, N., Gupta, S., Rusckowski,M., and Hnatowich, D. J. (2002) The influence of chain lengthand base sequence on the pharmacokinetic behavior of99mTc-morpholinos in mice. Q. J. Nucl. Med. 46, 233-243.

(34) Liu, G., He, J., Dou, S., Gupta, S., Vanderheyden, J. L.,Rusckowski, M., and Hnatowich, D. J. (2004) Pretargetingin tumored mice with radiolabeled morpholino oligomershowing low kidney uptake. Eur. J. Nucl. Med. Mol. Imaging31, 417-424.

(35) Zhang, Y. M., Liu, C. B., Liu, N., Ferro Flores, G., He, J.,Rusckowski, M., and Hnatowich, D. J. (2003) Electrostaticbinding with tat and other cationic peptides increases cellaccumulation of 99mTc-antisense DNAs without entrapment.Mol. Imaging Biol. 5, 240-247.

(36) Egholm, M., Buchardt, O., Christensen, L., Behrens, C.,Freier, S. M., Driver, D. A., Berg, R. H., Kim, S. K., Norden,B., and Nielsen, P. E. (1993) PNA hybridizes to complemen-tary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365, 566-568.

(37) Urtishak, K. A., Choob, M., Tian, X., Sternheim, N., Talbot,W. S., Wickstrom, E., and Farber, S. A. (2003) Targeted geneknockdown in zebrafish using negatively charged peptidenucleic acid mimics. Dev. Dynam. 228, 405-413.

(38) Good, L., and Nielsen, P. E. (1997) Progress in developingPNA as a gene-targeted drug. Antisense Nucleic Acid DrugDev. 7, 431-437.

(39) Rao, P. S., Tian, X., Qin, W., Aruva, M. R., Sauter, E. R.,Thakur, M. L., and Wickstrom, E. (2003) 99mTc-peptide-peptide nucleic acid probes for imaging oncogene mRNAs intumours. Nucl. Med. Commun. 24, 857-863.

(40) Gray, G. D., Basu, S., and Wickstrom, E. (1997) Trans-formed and immortalized cellular uptake of oligodeoxynucleo-

side phosphorothioates, 3′-alkylamino oligodeoxynucleotides,2′-O-methyl oligoribonucleotides, oligodeoxynucleoside me-thylphosphonates, and peptide nucleic acids. Biochem. Phar-macol. 53, 1465-1476.

(41) Soomets, U., Hallbrink, M., and Langel, U. (1999) Anti-sense properties of peptide nucleic acids. Front Biosci. 4,D782-D786.

(42) Basu, S., and Wickstrom, E. (1997) Synthesis and charac-terization of a peptide nucleic acid conjugated to a D-peptideanalogue of insulin-like growth factor 1 for increased cellularuptake. Bioconjugate Chem. 8, 481-488.

(43) Tian, X., Aruva, M. R., Qin, W., Zhu, W., Duffy, K. T.,Sauter, E. R., Thakur, M. L., and Wickstrom, E. (2004)External imaging of CCND1 cancer gene activity in experi-mental human breast cancer xenografts with Tc-99m-peptide-PNA-peptide chimeras. J. Nucl. Med. 45, 2070-2082.

(44) Boffa, L. C., Scarfi, S., Mariani, M. R., Damonte, G., Allfrey,V. G., Benatti, U., and Morris, P. L. (2000) Dihydrotestoster-one as a selective cellular/nuclear localization vector for anti-gene peptide nucleic acid in prostatic carcinoma cells. CancerRes. 60, 2258-2262.

(45) Tian, X., and Wickstrom, E. (2002) Continuous solid-phasesynthesis and disulfide cyclization of peptide-PNA-peptidechimeras. Organic Lett. 4, 4013-4016.

(46) Pallela, V. R., Thakur, M. L., Chakder, S., and Rattan, S.(1999) 99mTc-labeled vasoactive intestinal peptide receptoragonist: functional studies. J. Nucl. Med. 40, 352-360.

(47) Bartucci, M., Morelli, C., Mauro, L., Ando, S., and Surmacz,E. (2001) Differential insulin-like growth factor I receptorsignaling and function in estrogen receptor (ER)-positiveMCF-7 and ER-negative MDA-MB-231 breast cancer cells.Cancer Res. 61, 6747-6754.

(48) Thakur, M. L., Marcus, C. S., Saeed, S., Pallela, V.,Minami, C., Diggles, L., Le Pham, H., Ahdoot, R., andKalinowski, E. A. (2000) 99mTc-labeled vasoactive intestinalpeptide analogue for rapid localization of tumors in humans.J. Nucl. Med. 41, 107-110.

(49) Bieche, I., Laurendeau, I., Tozlu, S., Olivi, M., Vidaud, D.,Lidereau, R., and Vidaud, M. (1999) Quantitation of MYCgene expression in sporadic breast tumors with a real-timereverse transcription-PCR assay. Cancer Res. 59, 2759-2765.

(50) Jain, R. K. (1998) The next frontier of molecular medi-cine: delivery of therapeutics. Nat. Med. 4, 655-657.

(51) Thakur, M. L., Pallela, V. R., Consigny, P. M., Rao, P. S.,Vessileva-Belnikolovska, D., and Shi, R. (2000) Imagingvascular thrombosis with 99mTc-labeled fibrin alpha-chainpeptide. J. Nucl. Med. 41, 161-168.

(52) Lipponen, P., Eskelinen, M., and Syrjanen, K. (1995)Expression of tumour-suppressor gene Rb, apoptosis-sup-pressing protein Bcl-2 and c-Myc have no independentprognostic value in renal adenocarcinoma. Br. J. Cancer 71,863-867.

(53) de Jong, M., Rolleman, E. J., Bernard, B. F., Visser, T. J.,Bakker, W. H., Breeman, W. A., and Krenning, E. P. (1996)Inhibition of renal uptake of indium-111-DTPA-octreotide invivo. J. Nucl. Med. 37, 1388-1392.

BC0497923


Noninvasive Molecular Imaging of MYC mRNA Expression in Human Breast Cancer Xenografts with a [ 99m...

Documents

Transcript of Noninvasive Molecular Imaging of MYC mRNA Expression in Human Breast Cancer Xenografts with a [ 99m...