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Advances in Brief |
Department of Neuroradiology, University of Tübingen Medical School [S. H.], Central Section for Peptide Synthesis [R. P.], Division Biophysics of Macromolecules [W. W.], Division Organization of Complex Genomes [H. S.], Clinical Cooperation Unit Radiation Oncology [J. J., H. C-W., J. D., K. B.], and Central Section for Spectroscopy [C-W. v. d. L.], German Cancer Research Center, D-69120 Heidelberg, Germany
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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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Thus, microinjection methods were developed and initially used for administration of contrast media so that the Gd3+-complex could be accumulated in the intracellular space (1) . Other groups bound the Gd3+-complex to a carrier such as Motexafin (2) so that the Gd3+-complex could accumulate specifically in tumor cells. Finally, with the help of a viral carrier peptide (HIV-1 tat peptide; Ref. 3 ) and cationic lipids (4) , the Gd3+-complex could be accumulated nonspecifically in both tumor and nontumor cells.
However, these molecules show rapid influx and efflux characteristics. To achieve a longer stay of the Gd3+-complex in the cells we constructed, therefore, a Gd3+-complex bound to a PNA2
(5)
and connecting it to the amphiphilic transmembrane transport peptide (Fig. 1)
with the aim of observing the distribution of the Gd3+-complex. PNAs are peptidase- und nuclease-resistant modified oligonucleotides (6)
that are complementary to certain regions of the mRNA and hybridize in the cytoplasm.
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In normal cells, c-myc mRNA is hardly detectable. We used HeLa cells and the Dunning R3327 AT1 rat prostate adenocarcinoma in our study because the c-myc oncogene is particularly highly expressed in these cells (8) and this tumor (9) .
Until now, PNAs have been used in nuclear medicine (10 , 11) , but they have not yet been used in MRI. Our most important objective was to determine whether this contrast agent nonspecifically accumulated in the cytoplasm of both tumor and nontumor cells in vitro and in vivo.
Furthermore, we wanted to determine whether the PNA specificity for a tumor cell-expressed gene (here c-myc mRNA as an example) had any effect on the duration of contrast agent retention in the tumor cells.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Gd3+-Complex Formation
Stochiometric amounts of c-myc-specific complex-Lys-Lys-ATGCCCCTCAACGT-Cys-NH2 and gadolinium(III) chloride hexahydrate (Sigma-Aldrich, Taufkirchen, Germany; Cat. no. G7532) were dissolved in an aqueous NaCl solution (0.9%). After 12 h, the process was stopped. The procedure for forming the c-myc-nonspecific Gd3+-complex-Lys-Lys-GCCTAGACAATCTG-Cys-NH2 was identical.
Fluorochrome Labeling
All of the Gd3+-complex Lys-Lys-ATGCCCCTCAACGT-Cys-NH2 and the Gd3+-complex-Lys-Lys-GCCTAGACAATCTG-Cys-NH2 constructs of this study were FITC labeled only at the noncleavable lysine-spacer site on the 
-amino group.
Peptide Purification
All of the products were precipitated in ether and were purified by preparative HPLC (Shimadzu LC-8A, Duisburg, Germany) on a YMC ODS-A 7A S-7-µm reverse-phase column (20 x 250 mm), using 0.1% trifluoroacetic acid in water (eluent A) and 60% acetonitrile in water (eluent B). Peptides were eluted with a successive linear gradient, increasing from 25% to 60% B-eluent in 49 min at a flow rate of 10 ml/min. The fractions corresponding to the purified conjugate were lyophilized. Sequences of single modules as well as of the complete bimodular construct were characterized with analytical HPLC (Shimadzu, Duisburg, Germany LC-10) using a YMC-Pack Pro C18 (150 x 4.6-mm inner diameter) S-5-µm, 120A column with 0.1% trifluoracetic acid in water (eluent A) and 20% acetonitrile in water (eluent B). The analytical gradient ranged from 5% (eluent B) to 80% (eluent B) in 35 min (Fig. 1A)
. The constructs were further characterized with laser desorption mass spectrometry (Finnigan, Vision 2000; Fig. 1B
). Cysteine amide groups of the peptide TQVKIWFQNRRMKQKK-Cys-NH2 and the c-myc-specific PNA module Gd3+compound Gd3+-complex-Lys(FITC)Lys-ATGCCCCTCAACGT-Cys-NH2 were oxidized in the range of 2 mg x ml-1 in a 20% DMSO water solution. The reaction was completed 5 h later. The random sequence Gd3+-complex-Lys(FITC)Lys-GCCTAGACAATCTG-Cys-NH2 was linked under identical conditions. The progress of oxidation was monitored by analytical C18 reverse-phase HPLC.
Cell Culture
Exponentially growing human HeLa cervix carcinoma cells and nontumor cells (peripheral lymphocytes; German Cancer Research Center tumor bank) were cultivated in MEM culture medium (Sigma-Aldrich, Taufkirchen, Germany; no. #8028) supplemented with 10% fetal bovine serum (Sigma), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc., Karlsruhe, Germany). Cells were grown as monolayers in a mycoplasm-free state as monitored by PCR (Mycoplasma PCR Primer Set; Stratagene, Amsterdam, The Netherlands).
Cell Viability
Cell viability was assessed by dye exclusion assay. HeLa cells and lymphocytes were incubated with both conjugates (c-myc-specific and -nonspecific Gd3+-complex; 0.5 mM) for 24, 48, and 72 h. Nontreated cells served as controls for the same time periods. Five min after trypan blue staining (0.4%), viable and nonviable cells were microscopically quantified (Fig. 2)
. Cell counts for each experimental series were repeated five times.
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Biological variation of transplanted tumors was minimized by applying the following procedures. For each animal, we always used tissue specimens from one tumor of a donor animal. This tumor, in turn, was obtained by transplanting tumor tissue from a cryopreserved stock to the donor animal. Fresh specimens (2 x 2 x 2 mm3) of tumor tissue were transplanted s.c. into the left thighs of young male adult Copenhagen rats (Charles River Sulzbach Germany). MRI was performed 3 weeks after transplantation, when the tumor volume was 1–2 cm3. All of the procedures were carried out under anesthesia induced with Ketavet (Parke Davis, Morris Plains, NJ; 0.9 g/kg i.p.) and Rompun 2% solution (Bayer, Leverkusen, Germany; 0.3 g/kg i.p.) and maintained by inhalation of 0.5% halothane in a mixture of 40% nitrous oxide (Hoechst, AG, Frankfurt/Main, Germany) and 60% oxygen (Messer Griesheim GmbH, Frankfurt/Main, Germany).
All animal experiments were approved by an external animal protection committee. The rats were kept two per cage under controlled temperature (24 ± 1°C), humidity (50 ± 10%), and light (12-h light/dark cycle) conditions with free access to food and water, according to the guidelines for laboratory animals established by the German Government.
Intracellular Localization in Vitro
To perform fluorescence microscopic studies, we plated HeLa cells and lymphocytes (5 x 105) on sterile, silane-coated glass slides embedded in quadriPERM plus (Heraeus, Hanau, Germany) and incubated them for 24 h. After two wash cycles with MEM, the cells were incubated with the FITC-labeled c-myc-specific Gd3+-complex (100 pM) at 37°C in a 5% CO2 atmosphere for 30 min. The culture medium was removed, and the cells were washed twice so that microscopic studies could be performed later. The intracellular distribution of the FITC-labeled constructs was verified using a Zeiss Laser confocal microscope (LSM 510 UV). For excitation of the FITC, a filter set with 488- and 522-nm emission filters was used. The optical slice thickness was 700 nm. The excitation line of an argon ion laser was used to detect a fluorescence signal from the FITC-labeled c-myc-specific Gd3+-complex. To increase the contrast of the optical sections, 12–20 single exposures were averaged. The image acquisition parameters were adapted to show signal intensities in accordance with the visible microscopic image. The same experiments were performed with a random sequence Gd3+-complex construct.
Generation of Three-Dimensional Molecular Models
To learn more about the spatial requirements of the investigated bioconjugates, we generated and, subsequently, manually connected three-dimensional molecular models of the basic molecular modules (TPU, PNA, and Gd3+-complex; Fig. 3
). Because no experimental three-dimensional structures of the peptide modules were available, spatial models were generated based on homologous data. The FASTA search option of the Protein Data Bank (16)
was used to identify sequences that show a high similarity to the TPU (TQVKIWFQNRRMKQKK-Cys-NH2) and the c-myc-specific PNA (ATGCCCCTCAACGT-Cys-NH2). For TPU, the crystal structure of the site-specific recombinase XerD (PDB 1A0P: 217–231 sequence QMTRQTFWHRIKHYA) was taken as a template, for which an 85% identity in a 13-amino-acid overlap was shown. The biopolymer option of the INSIGHTII module was applied to mutate the required amino acids. The construct was then minimized, followed by a short molecular dynamics simulation in aqueous solution to relax the model. The AMBER force field was used to accomplish this procedure. The PNA model was based on the crystal structure of the PNA-DNA complex (PDB-entry 1PNN). The nucleotides were changed manually according to the sequence ATGCCCCTCAACGT-Cys-NH2. The three-dimensional structure of the Gd3+-complex was taken from the Cambridge Structural Database (entry heqbua; Ref. 17
). Finally, the aforementioned molecular modules were connected manually in such a way that a spherically favorable orientation was achieved. The final construction of the bioconjugate was accomplished using the INSIGHTII software (Accelrys, San Diego, CA), which was also used to produce the graphical representations of the bioconjugates. Although it is clear that the constructed spatial structures of the bioconjugate show only one of a multitude of possible conformations that peptides, which are flexible molecules, can exhibit, they represent to some extent a realistic spatial model; that is, an all-atom model of the entire molecule is presented that has the correct bond length and bond angle. Consequently, the relative size of the connected peptide modules and their spatial shape are realistic and can be directly compared. The physicochemical properties, which can be mapped onto the molecular surface, reflect realistic electronic characteristics of the molecule.
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For MRI, a 1.5-T whole-body Siemens Magnetom Vision Plus with a standard circular polarized head coil was used. The test tubes were firmly positioned parallel to each other totally submerged in a water bath. The imaging protocol consisted of a sagittal and axial T1-weighted spin-echo sequence (TR: 600ms/TE:15 ms; scan time, 45 s). The FOV was 200 mm x 200 mm, using a 256 x 256 imaging matrix and two acquisitions. Slice thickness was 2 mm, resulting in a pixel size of 0.79 mm x 0.78 mm.
The T1 and T2 relaxation times within the pellets of the three tubes (c-myc-specific Gd3+-complex, c-myc-nonspecific Gd3+-complex, Magnevist) were measured to evaluate the intracellular relaxivity of the respective contrast agents (r = 1/T1). The T1 relaxation time was measured by an inversion recovery sequence (TR: 5000 ms/TE: 76 ms/TI: 25–4000 ms; 15 different TI values, scan time, 15 x 25 s; FOV, 160 mm x 160 mm; matrix, 132 x 256; slice thickness, 7 mm; pixel size, 1.21 x 0.63 mm). T2 relaxation time was measured by a multiecho sequence (TR; 5000 ms/16 TE values, 30–245 ms; FOV, 250 mm x 250 mm; matrix, 256 x 256; slice thickness, 5 mm; pixel size, 0.98 x 0.98 mm; scan time, 21 min 21 s). Signal intensity measurements were obtained from HeLa cervix carcinoma cells and the background. A tube with HeLa cells, incubated in MEM without contrast agent, was used as a control.
Efflux of the c-myc-specific and -nonspecific Gd3+-Complex in HeLa Cells.
Because of a signal intensity maximum in HeLa cells after a 60-min incubation period (c-myc-specific Gd3+-complex) and 30-min incubation period (c-myc-nonspecific Gd3+-complex), we decided to begin the efflux measurements after 30 and 60 min, respectively. After this period, the cells were washed with conjugate-free MEM to remove all traces of the Gd3+-complex. This procedure was repeated hourly until no signal increase compared with the control tube (HeLa cells in MEM without contrast agent) could be detected in T1-weighted sequences. All of the influx and efflux experiments were performed three times.
Influx and Efflux of the c-myc-specific and -nonspecific Gd3+-Complex in Lymphocytes.
The same uptake and efflux experiments were conducted using lymphocytes as were performed with HeLa cells.
Kinetic Studies in Copenhagen Rats in Vivo.
All of the MRI in vivo studies were carried out by using a 1.5 T clinical MRI Siemens Magnetom Vision with the tumor-bearing rats (n = 7) in prone position in a standard circular polarized head coil. The imaging protocol consisted of a coronal and axial T1-weighted spin-echo sequence (TR: 600ms/TE:15 ms; scan time, 45 s). The FOV was 200 mm x 200 mm, using a 256 x 256 imaging matrix and two acquisitions. Slice thickness was 2 mm, resulting in a pixel size of 0.79 mm x 0.78 mm. All of the procedures were carried out as described in the section above "Animal Model and Tumor System". Non-contrast-enhanced imaging studies were first performed in all of the rats (n = 7).
Then, both the c-myc-specific and -nonspecific contrast agents were administered in an aqueous solution (0.1 ml, 0.25 µmol/kg). In a second trial, signal intensity measurements were obtained from Dunning R3327 AT1 rat prostate adenocarcinoma cells.
Influx and Efflux of Both the c-myc-specific and -nonspecific Contrast Agent from the Tumor.
After the non-contrast-enhanced study was finished, the contrast agent with the c-myc-nonspecific PNA was administered via the tail vein and the influx and efflux in the AT1 tumor were observed until the MRI signal disappeared.
In the same rat, the native non-contrast-enhanced study was performed as a control 18 h later, but the contrast agent with the c-myc-specific PNA was administered. Influx and efflux in the tumor were observed as above.
Correlation between MRI Signal Intensity with Intracellular Localization of the FITC-labeled c-myc-specific and -nonspecific Gd3+-Complex in CLSM, 20 and 300 min after i.v. Injection.
Two rats were first examined without the contrast agent. As a control, the c-myc-nonspecific contrast medium was then injected i.v. into the tail vein.
The first of the two rats was killed 20 min after the injection, the second after 300 min. Then the tumor, organs (lung, liver, brain, spleen, and kidneys) and muscle tissue were excised and were frozen in isopentane (-20C°) for 10 min. The intracellular distribution of the FITC-labeled c-myc-nonspecific constructs were documented with a CLSM (510 UV; Carl Zeiss, Jena, Germany). For the fluorescence excitation of the FITC-labeled c-myc-nonspecific Gd3+-complex, we used the 488-nm line of an argon ion laser and appropriate beam splitters and barrier filters. The confocal aperture diameter was adjusted for an optical slice thickness of 700 nm. The image acquisition parameters were adapted as described in the in vitro imaging section.
In addition, a non-contrast-enhanced MRI study was performed in two rats, and then the c-myc-specific contrast agent was administered i.v. After 20 min, the first rat was killed; and after 300 min, the second rat was killed, and the tumor and organs were examined by CLSM, as described above.
Before the animals were killed at the times indicated above, an MRI study was performed.
Biostatistical Evaluation
The enhancement curve was estimated using a nonlinear regression method. To estimate the course of enhancement curve precisely enough, the sample of at least four animals shall be used. The averaging of four measurements at each time point leads to the reduction of SD by 50%. Therefore, in our study, a total of seven animals were examined (18)
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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In MRI, an increased intracellular signal intensity in HeLa cells could be detected after just 10 min of incubation with the c-myc-specific Gd3+-complex and subsequently reached a maximum after 1 h (whole body 1.5 T Siemens Magnetom, standard circular polarized head coil; Fig. 4A
). This result was confirmed using CLSM. By way of comparison, if Gd3+-DTPA (Magnevist) alone was used as a contrast agent, the signal change was no stronger than that in the HeLa cells that had been incubated in MEM alone. The measured relaxivity R within the HeLa cell pellets changed by more than a factor of 3 after incubation with the c-myc-specific Gd3+-complex (R = 0.000314) as compared with that after incubation with Gd3+-DTPA alone (R = 0.000995).
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The intracellular localization of the contrast agent in the tumor, and in all other organs as well, was confirmed by CLSM (Fig. 5)
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In vivo autoradiographic studies in rats have already shown that an iodinated contrast agent with a PNA against luciferase mRNA was retained longer in the cytoplasm of cells with high levels of luciferase mRNA than in cells lacking luciferase mRNA (10) .
Whether the gadolinium complex could be specifically targeted against certain mRNA sections because of the PNA binding and could be retained longer in these cells and visible on MRI because of the hybridization has not yet been studied.
With respect to the effect of PNA specificity for the c-myc mRNA on the duration of contrast agent retention in the tumor cells, we found that, both in vitro (HeLa cells) and in vivo (prostate adenocarcinoma), the gadolinium contrast agent was retained in the tumor cells for any length of time only if the c-myc-specific PNA was used (HeLa cells in vitro, about 4 h; prostate adenocarcinoma in vivo, more than 5 h; Figs. 4, A and C
, and 5
). Expression levels of c-myc mRNA are high in the cytoplasm of Dunning R3327 AT1 rat prostate adenocarcinoma and HeLa cells (8
, 9)
.
Using a random sequence, we saw rapid efflux out of tumor cells (Figs. 5, A and C)
. This suggests specificity of the c-myc PNA. In nontumor cells (e.g., lymphocytes and lung, muscle, brain, hepatic, splenic, and muscle cells), showing only low expression of the c-myc mRNA, a rapid efflux of the contrast agent was observed for both the c-myc-specific and the c-myc-nonspecific PNA (Figs. 4B
and 5
).
One might speculate that the slower efflux of the c-myc-specific gadolinium complex from the tumor cells is possibly caused by a certain property of the tumor cell membrane as compared with the membranes of healthy cells. The longer retention of the c-myc-specific contrast agent would then be independent of PNA design. However, an argument against this, although not proof of it, is the rapid efflux of the random sequence contrast agent out of the tumor cells.
In the animal experiments, the uptake and efflux of the c-myc-nonspecific and -specific gadolinium contrast agents were tested subsequently in Dunning R3327 rat prostate adenocarcinoma to establish the same baseline conditions. Using this method, we could avoid the situation of testing one contrast agent in a well-vascularized tumor and another in a poorly vascularized, necrotic tumor, which would produce different results.
The question remains as to whether the c-myc-nonspecific contrast agent, initially given i.v., affects the baseline conditions for the subsequently administered c-myc-specific contrast agent. The second (c-myc-specific) contrast agent was administered at a point when an increase in intensity of the MRI signal could no longer be registered. This was also shown using CLSM. If, however, fresh HeLa cells are incubated in vitro with the two contrast agents, the same uptake and efflux characteristics are seen as for the Dunning AT1 tumors in vivo.
To summarize, there is considerable indication that this particular gadolinium contrast agent can be used to demonstrate the presence of tumors and possibly metastases. However, we are still investigating whether this applies to more, or even all, tumors. One issue that is still completely unclear is whether the transient intracellular accumulation of the contrast agent causes cell damage, although the cell growth was not affected according to results of viability tests.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Clinical Cooperation Unit Radiation Oncology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Phone: 49-6221-422495; Fax: 49-6221-42-52-2495; E-mail: k.braun{at}dkfz.de 
2 The abbreviations used are: PNA, peptide nucleic acid; MRI, magnetic resonance imaging; HPLC, high-performance liquid chromatography; TPU, transmembrane transport unit; DTPA, diethylenetriaminepentaacetic acid; FOV, field of view; CLSM, confocal laser scanning microscope/microscopy.
Received 4/11/03. Revised 5/30/03. Accepted 7/31/03.
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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