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[Cancer Research 63, 7221-7231, November 1, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Switching Off HER-2/neu in a Tetracycline-Controlled Mouse Tumor Model Leads to Apoptosis and Tumor-Size-Dependent Remission1

Ilka B. Schiffer2, Susanne Gebhard, Carolin K. Heimerdinger, Annette Heling, Jochem Hast, Ursula Wollscheid, Barbara Seliger, Berno Tanner, Sandra Gilbert, Thomas Beckers, Silke Baasner, Walburgis Brenner, Christian Spangenberg, Dirk Prawitt, Tatjana Trost, Wolfgang G. Schreiber, Bernhard Zabel, Manfred Thelen, Hans-Anton Lehr, Franz Oesch and Jan G. Hengstler

Institute of Toxicology [I. B. S., S. Ge., C. K. H., F. O.], Departments of Radiology [A. H., J. H., W. G. S., M. T.], Hematology [U. W., B. S.], and Gynaecology [B. T.], University of Mainz, 55131 Mainz; Baxter Oncology GmbH, 60314 Frankfurt [S. Gi.]; Atlanta Pharma, 78467 Konstanz [T. B.]; Zentaris, 60314 Frankfurt [S. B.]; Urological Clinic and Polyclinic [W. B.], Children’s Hospital [C. S., D. P., T. T., B. Z.], and Institute of Pathology [H. A. L.], University of Mainz, 55131 Mainz; Institute of Legal Medicine and Rudolf-Boehm Institute of Pharmacology and Toxicology, Center for Toxicology, University of Leipzig, 04107 Leipzig [J. G. H.], Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 Immunohistochemistry
 Flow Cytometry
 Statistical Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
Overexpression of the receptor tyrosine kinase HER-2/neu is associated with poor prognosis in patients with breast and ovarian cancer. Recent excitement has surrounded the therapeutic effects of HER-2-blocking therapy strategies and has rekindled interest on the molecular mechanisms of HER-2/neu in tumor biology. To study the role of HER-2/neu overexpression in vivo, we used a murine fibroblast cell line (NIH3T3-her2) conditionally expressing human HER-2/neu under control of a tetracycline-responsive promoter. Expression of HER-2 could be down-regulated below detection limit (>625-fold dilution) by exposure of NIH3T3-her2 cells to anhydrotetracycline (ATc). Subcutaneous injection of NIH3T3-her2 cells into nude mice resulted in rapid tumor growth. Mice with mean tumor volumes of 0.2, 0.8, 1.9, and 14.9 cm3 were treated daily with 10 mg/kg ATc to switch off HER-2/neu expression, producing reductions in tumor size of 100, 98.1, 81.4, and 74.2%, respectively, by 7 days after onset of ATc administration (P = 0.005, Kruskal–Wallis test). Different long-term effects of HER-2 down-regulation were observed when mice with small (0.2 cm3; n = 7), intermediate (0.8–1.2 cm3; n = 10) and large (>=1.9 cm3; n = 11) tumors received ATc for up to 40 days. Complete remission was observed for 100, 40, and 18% of the small-, intermediate-, and large-sized tumors, respectively (P = 0.003). However, after 20–45 days of ATc administration, recurrent tumor growth was observed for all mice, even in those with previous complete remissions. The time periods for which mean tumor volume could be suppressed to volumes <0.1 cm3 under ATc administration were 34, 22, 8, and 0 days for tumors with initial volumes of 0.2, 0.8, 1.9 and 14.9 cm3, respectively (P = 0.005, Kruskal–Wallis test). Interestingly, HER-2 remained below the detection limit in recurrent tumor tissue, suggesting that initially HER-2-dependent tumors switched to HER-2 independence. The "second hits" leading to HER-2-independent tumor growth have not yet been identified. The rapid regression of tumors after down-regulation of HER-2 was explained by two independent mechanisms: (a) a block in cell cycle progression, as evidenced by a decrease in Ki-67 antigen expression from 40% before ATc treatment to 8.3% after 7 days of ATc treatment; and (b) induction of apoptosis as demonstrated by caspase-3 activation and by the terminal deoxynucleotidyltransferase (Tdt)-mediated nick end labeling assay (TUNEL). In conclusion, we have shown that switching off HER-2 may disturb the sensitive balance between cell proliferation and cell death, leading to apoptosis and tumor remission. Tumor remission was dependent on the volume of the tumors before down-regulation of HER-2/neu.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 Immunohistochemistry
 Flow Cytometry
 Statistical Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
The epidermal growth factor receptors (HER/erbB) constitute a family of four members involved in cell cycle control and cell differentiation (1 , 2) : (a) HER-1, also termed epidermal growth factor; (b) HER-2, also termed neu or erbB-2; (c) HER-3, also termed erbB-3; and (d) HER-4, also termed erbB-4. Ligand binding to HER-1, -3, or -4 induces receptor dimerization in a sequential ligand-induced receptor dimerization process (3 , 4) . HER-2/neu can heterodimerize with HER-1, -3, or -4 to generate more effective intracellular signals than either the epidermal growth factor receptor or the HER-2/neu homodimers alone (1 , 4 , 5) . HER-2/neu activation induces several signal transduction pathways, such as (a) the ras/MAP3 kinase pathway, which results predominantly in cell cycle progression (6 , 7) ; (b) phosphatidylinositol 3,4,5-trisphosphate kinase/AKT pathway (7, 8, 9) , which leads to antiapoptotic signaling; and (c) phospholipase C{gamma} (PLC; Ref. 10 ), which interacts with the ras/MAP kinase pathway (Fig. 1A)Citation . In addition to its role in apoptosis and proliferation, there exists evidence that HER-2/neu might also mediate immune escape mechanisms (11) .



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  		Fig. 1. A, HER-2-mediated signal transduction. ERK, extracellular signal-regulated kinase; I{kappa}B, inhibitor of nuclear factor-{kappa}B; Mek, MAP kinase kinase; NF-{kappa}B, nuclear factor-{kappa}B; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PKB, protein kinase B; B, generation of the tetracycline-controlled cell line NIH3T3-her2 by cotransfection of NIH3T3 cells with plasmids pUHD15-1 (22) , pTBC1 Hygro, and pTBC HER2/SEAP (21) . pUHD15-1 expresses the tetracycline-controlled transactivator tTA, which stimulates expression of HER-2/neu from the vector pTBC HER2/SEAP and expression of a hygromycin resistance gene from the vector pTBC1 Hygro. Tetracycline inactivates the transactivator protein tTA, leading to down-regulation of HER-2 and loss of hygromycin resistance (TET-OFF system).

 
Gene amplification and/or overexpression of HER-2are found in up to 30% of human breast and ovarian carcinomas (1 , 12) , as well as in other malignant tumors (13) . Overexpression of HER-2is associated with adverse prognosis, which has been linked with increased invasiveness and/or decreased response to chemotherapy (12) . Recent excitement has surrounded the clinical development and remarkable therapeutic effects of novel treatments that target HER-2/neu, such as trastuzumab (Herceptin; Refs. 2 , 14, 15, 16, 17, 18, 19, 20 ).

To analyze the molecular mechanisms involved in HER-2/neu-blocking therapy in vivo, we used a model system of murine fibroblasts conditionally expressing human HER-2/neu under the control of a tetracycline-responsive promoter (NIH3T3-her2; Ref. 21 ). Subcutaneous. injection of NIH3T3-her2 cells into the dorsal skin of nude mice resulted in growth of solid tumors. We show here that switching off HER-2/neu expression by treatment with ATc causes size-dependent tumor remission, which is associated with a decrease in cell cycle progression and induction of apoptosis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
Immunohistochemistry
 Flow Cytometry
 Statistical Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Cell Culture
NIH3T3, an immortalized cell line originally derived from mouse embryo fibroblasts, was obtained from American Type Culture Collection. Wild-type NIH3T3 and its derivatives were grown in DMEM (PAN, Aidenbach, Germany) supplemented with 10% fetal bovine serum (tetracycline free; Clontech, Palo Alto, CA) and 1% penicillin–streptomycin (PAN). Cells were cultured at 37°C in 5% CO2 humidified air. Conditional expression of HER-2 was achieved by using of the TET-OFF system originally described by Gossen et al. (22) . Briefly, wild-type NIH3T3 cells were cotransfected with three vectors (pUHD 15-1, pTBC1 Hygro, and pTBC HER2/SEAP; Fig. 1BCitation ) as described by Baasner et al. (21) , resulting in a cell line termed NIH3T3-her2. Cotransfection led to tetracycline-controlled HER-2 expression. Exposure of cells to ATc led to complete down-regulation of HER-2 (21) . Selection for stable transfection was achieved by adding 125 µg/ml hygromycin B (Sigma–Aldrich, Schnelldorf, Germany) to the cell culture medium. Expansion of NIH3T3-her2 cells was performed in the presence of hygromycin B. In contrast, all experiments including exposure to ATc were done in the absence of hygromycin B.

In Vitro Inhibition of HER-2/neu Expression
NIH3T3-her2 cells were harvested from 90% confluent cultures in dishes and plated at a density of 105 cells/dish on 75-cm2 flasks. For down-regulation of HER-2/neu, NIH3T3-her2 cells were incubated with 10 ng/ml ATc (Acros Chimica, Geel, Belgium) for 0 (controls), 1, 3, and 7 days. ATc was added 24 h after plating of cells. For all four incubation periods with ATc, cells were cultured for 8 days and harvested simultaneously. The design of the study was to start incubations with ATc at different time points but to harvest all dishes simultaneously. For the immunohistochemical analysis, cells were cultured on SUPERFROST PLUS slides (Menzel-Gläser, Braunschweig, Germany). The slides were air dried overnight and stored at -20°C until immunohistochemical analysis.

Determination of Tumor Growth
We injected 7 x 106 NIH3T3-her2 cells s.c. into the dorsal skin of 3–4 week-old male nude mice (cd nu-/nu-; Charles River, Sulzfeld, Germany). Animals were housed under specific pathogen-free conditions. Eight to 10 days after injection of the NIH3T3-her2 cells, small tumors with a mean diameter of 0.5 cm became visible. The tumor diameter was measured with a caliber rule. The maximum and minimum diameters of the tumor were determined. The mean value of the maximum and minimum diameters was defined as the mean diameter. For Western blot analysis and immunohistochemistry, tumors were treated for 1, 3, and 7 days with 10 mg ATc/kg of body weight. Thereafter, mice were sacrificed by cervical dislocation. Tumors were isolated, shock-frozen in liquid nitrogen-cooled 2-methylbutane, and stored at -80°C. Mice not receiving ATc therapy were used as negative controls. Each time an ATc-treated tumor was harvested, a time-matched control tumor was harvested simultaneously. Control tumors continually increased in size to a mean diameter of 3.5 cm at which time mice were sacrificed. Tumor volume (V) was calculated by the formula: V = a · b · b/2, where a represents the minimum and b the maximum tumor diameter. Complete remission was achieved when absolutely no tumor volume was visible macroscopically. The treatment protocols, including the groups of mice with different tumor volumes, are shown in Table 1Citation . To study the possibility of achieving a complete remission, we tested additional mice, giving 7, 10, and 11 mice with initial tumor volumes of 0.2, 0.8–1.2, and >=1.9 cm3, respectively.


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  		Table 1 Treatment protocols of nude mice after induction of subcutaneously growing NIH3T3-her2 tumors

 
MRI
MRI measurements were performed as described by Hast et al. (23) with use of a 1.5-Tesla whole-body MR-System (Magnetom Experimental; Siemens, Erlangen, Germany). Mice were anesthetized by s.c. injection of 61.5 mg/kg ketamine (Ketamine–ratiopharm 50; Ratiopharm, Ulm, Germany) and 2.3 mg/kg xylazine (Rompun 2%; Bayer, Lerverkusen, Germany). After a bolus injection of saline-diluted contrast agent (Magnevist; Schering AG, Berlin, Germany) into the tail vein, T1-weighted sequences were performed.

Western Blot Analysis
Preparation of Cell Extracts.
Cell cultures were scraped from culture dishes, washed twice in PBS, and resuspended in solubilization buffer. Tumor tissue was pulverized by use of a mortar and pestle on dry ice and then lysed in solubilization buffer by sonification (three times 10 strokes; Labsonic U; B. Braun Medical AG, Emmenbrücke, Germany) on ice. To remove cell debris, the suspensions were centrifuged at 18,000 x g for 10 min. The supernatants were collected, and protein concentrations were determined by the Bradford assay (24) . Two types of solubilization buffers were used. For preparation of HER-2/neu membrane proteins, a lysis buffer was used, containing 25 mM Tris-phosphate, 2 mM EDTA, 2 mM DTT, 10% glycerol, and 1% Triton-X-100 (pH 8). For solubilization of nuclear proteins such as caspase-3, a CHAPS cell extraction buffer was used, containing 20 mM PIPES (adjusted with NaOH to pH 7.2; Amersham Life Science, Cleveland, OH), 0.1% CHAPS, 10 mM DTT, 100 mM NaCl, 1 mM EDTA, and 10% sucrose. Both solubilization buffers were supplemented with 1% of a commercially available protease inhibitor cocktail (Sigma) containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), bestatin, leupeptin, and aprotinin.

Western Blotting.
Total cellular proteins (25 µg for analysis of HER-2 and 40 µg for analysis of caspase-3) were mixed with sample buffer according to the protocol of Laemmli (25) and resolved on a 10% (HER-2) or 15% (caspase-3) SDS–polyacrylamide gel by electrophoresis. Thereafter, proteins were electrotransferred to Poly Screen polyvinylidene difluoride transfer membranes (NEN Life Science, Boston, MA). The membranes were blocked with PBS-Tween (PBS + 0.1% Tween 20) containing 10% Roti-Block (Roth, Karlsruhe, Germany) for 1 h and then incubated with the anti-HER-2/neu or anti-caspase-3 antibodies for 2 h or, as a loading control, with the anti ß-actin antibody for 30 min at room temperature. After washing, the membranes were incubated with the secondary antibody, horseradish peroxidase-conjugated antimouse immunoglobulin for 20 min or antirabbit immunoglobulin for 40 min at room temperature. After a final wash, proteins were visualized with a chemiluminescence detection system (Western Lightning Chemiluminescence Reagent Plus; Perkin-Elmer Life Science, Boston, MA) with subsequent exposure to Kodak X-OMAT films. The expression levels were quantified by densitometric analysis using Scion Imaging software (Scion Image ß 4.02 for Windows 98). The MagicMark Western Standard from Invitrogen GmbH (Karlsruhe, Germany) served as an internal protein standard. For repeated staining, individual membranes were stripped with a buffer containing 0.76% Tris base, 2% SDS, and 0.7% 2-mercaptoethanol adjusted with HCl to pH 6.8 for 1 h at 50°C. The SDS–polyacrylamide gel and the membrane (after the last staining with antibodies) were also stained with Coomassie blue to confirm that equal amounts of proteins had been applied to each lane.

Origin and Dilution of Antibodies.
The monoclonal antibody against human HER-2/neu (185-kDa protein) was obtained from Quartett (Berlin, Germany) and used at a dilution of 1:270. The monoclonal antibody against mouse ß-actin (42-kDa protein; Sigma) was used at a dilution of 1:2000. The antimouse caspase-3 antibody (Cell Signaling; Biolabs, Frankfurt, Germany), detects the full length caspase-3 (32–35 kDa) and the cleaved large fragment (17–20 kDa) of caspase-3. The anti-caspase-3 antibody was diluted 1:500. The secondary antibody, peroxidase-linked antirabbit, was obtained from Cell Signaling (Biolabs) and used in a dilution of 1:1500. Peroxidase-linked antimouse antibody (Sigma) was used at a dilution of 1:5000 to detect ß-actin and at 1:50,000 to detect HER-2. Primary and secondary antibodies were all diluted in PBS-Tween containing 10% Roti-Block.


   Immunohistochemistry
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Immunohistochemistry
Flow Cytometry
 Statistical Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Cryosections.
Using a cryotome (CM 3000 Cryostat; Leica Instruments GmbH, Nussloch, Germany), we produced 5-µm-thick cryosections and transferred them to SUPERFROST PLUS slides. After air drying overnight, slides were stored at -20°C until use.

Immunohistochemical Detection of HER-2/neu.
The frozen slides were thawed to room temperature, fixed in cold (-20°C) acetone for 10 min, and rinsed for 10 min in TBS [50 mM Tris, 150 mM NaCl (pH 7.4–7.5)]. To block endogenous peroxidase, slides were rinsed in methanol containing 8.5% H2O2 for 30 min at room temperature and washed three times in TBS for 5 min each. Subsequently, nonspecific binding of immunoglobulin was blocked by the addition of 3% BSA in TBS for 1 h at room temperature. The primary monoclonal antibody against HER-2/neu (rabbit antihuman HER-2, a ready-to-use solution; DAKO, Hamburg, Germany) was incubated for 30 min at room temperature. For detection of the primary antibody, we used a commercially available detection kit (ABC-kit, XHCO2; Dianova, Hamburg, Germany). The slides were washed three times in TBS and incubated with biotinylated goat antirabbit antibodies (diluted 1:50) for 30 min at room temperature (Dianova). After slides were rinsed in TBS, the ABC-Reagent (avidin and biotinylated horseradish peroxidase) was added for 30 min at room temperature. The slides were then washed again with TBS. The 3,3'diaminobenzidine solution (0.6% 3,3'diaminobenzidine–0.3% H2O2 in H2O) was added for 5 min. The slides were then rinsed for 10 min under tap water. Counterstaining was performed in hematoxylin solution (1:10 dilution in distilled water; Merck, Darmstadt, Germany) for 1.5 min, after which the slides were again rinsed for 10 min under tap water. To obtain stronger contrast of the hematoxylin stain, slides were placed in bluing reagent (70% ethanol containing 1.5% NH4OH) for 1 min followed by rinsing in tap water. The tissue was dehydrated by incubation with increasing alcohol concentrations (70, 96, 100, and 100% isopropanol for 10 s each). After being rinsed in Rotihistol (Roth, Karlsruhe, Germany), the slides were covered with Entellan Neu (Merck). All slides were processed in one session. Two slides obtained from the same tumor were used as positive and negative controls. The negative control was prepared by use of TBS in place of the primary antibody.

Immunohistochemical Detection of Ki-67.
In addition to the staining with the antihuman HER-2 antibody, the same protocol was also performed with an antimouse Ki-67 antibody (Ki-67 rabbit antimouse; Dianova, Hamburg, Germany) diluted 1:50 in TBS containing 5% FCS.

TUNEL Assay.
For detection of DNA strand breaks in individual cells by light microscopy, a commercially available kit (In Situ Cell Death Detection Kit, POD; Roche Diagnostics GmbH, Mannheim, Germany) was used. A modified TUNEL protocol was performed. The frozen slides were fixed directly with 1% paraformaldehyde (pH 7) for 30 min at room temperature (26) and washed with TBS for 10 min. Thereafter, slides were rinsed in TBS containing 0.1% Triton-X-100 and 0.1% sodium citrate for 2 min at 4°C. For the following staining procedure, the same protocol was used as described for immunohistochemical detection of HER-2/neu. The TUNEL reaction mixture and the converter–POD solution were prepared according to the manufacturer’s instructions.

Evaluation of Immunohistochemical Slides.
Using the antihuman HER-2/neu antibody, the antimouse Ki-67 antibody, and the TUNEL reagent, we determined the percentage of HER-2/neu-, Ki-67-, and TUNEL-positive tumor cells in relation to all tumor cells. For this purpose, five representative areas with vital tumor cells or tissue were randomly selected. Evaluation was performed with a Nikon Optiphot microscope with x400 magnification. The percentage of HER-2/neu (cytoplasmic and membrane)-positive cells that were also positive for Ki-67 and TUNEL (nuclear) staining was determined independently by two experienced investigators (J. G. H. and I. B. S.). Mean values for all five areas were calculated. In all cases the values obtained by both investigators differed by <10%.


   Flow Cytometry
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Immunohistochemistry
 Flow Cytometry
Statistical Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
We used 1 x 106 NIH3T3-her2 cells for staining with the mouse antihuman monoclonal antibody directed against the extracellular domain (Ab-5; Oncogene, Darmstadt, Germany) and the mouse antihuman monoclonal antibody directed against the intracellular, COOH-terminal domain of human HER-2/neu (Ab-3; Oncogene). Staining with the monoclonal antibody directed against rat HER-2/neu (Ab-4; Oncogene) served as negative control. Cells were incubated with the respective antibodies for 20 min at 4°C and washed twice before FITC-conjugated antimouse IgG (DTAF; Dianova) was added as a secondary antibody. After samples were washed with PBS, they were analyzed by flow cytometry (Coulter EPICS XL; Beckman Coulter, Krefeld, Germany), using the Expo 32 software (Beckman Coulter). Before incubation with Ab-3, directed against the intracellular domain of HER-2, cells were permeabilized with the IntraPrep Permeabilization Reagent (Immunotech, Marseilles, France). The results are expressed as mean specific fluorescence intensities.

All chemicals and reagents not specifically mentioned were obtained from Sigma-Aldrich (Schnelldorf, Germany).


   Statistical Analysis
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Immunohistochemistry
 Flow Cytometry
 Statistical Analysis
RESULTS
 DISCUSSION
 REFERENCES
 
For statistical analysis of possible differences in the occurrence of complete remission of tumors with different volumes, we used the {chi}2 test (two-sided). The Kruskal–Wallis test (two-sided) was applied to test for (a) differences in the extent of tumor remission between groups of mice with different tumor volumes at the onset of ATc administration, and (b) differences in the time periods for which mean tumor volumes could be suppressed to volumes <=0.1 cm3 between groups of mice with different tumor volumes at the onset of ATc administration.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Immunohistochemistry
 Flow Cytometry
 Statistical Analysis
 RESULTS
DISCUSSION
 REFERENCES
 
Conditional Expression of HER-2/neu in Vitro.
Human HER-2 was conditionally expressed in NIH3T3 cells as described by Baasner et al. (21) . The TET-OFF system based on a tetracycline-controlled transactivator protein, tTA (22) , was used (Fig. 1B)Citation . The resulting cell line, named NIH3T3-her2, was incubated with ATc (10 ng/ml) for 1, 3, 7, and 9 days. A clear decrease in HER-2/neu expression was observed during incubation with ATc as evidenced by Western blot analysis (Fig. 2A)Citation . Results similar to those shown in Fig. 2ACitation were obtained in three independent experiments. The resulting mean (±SD) HER-2 expression values obtained by densitometric analysis were 50.8 ± 12.3%, 24.0 ± 15.3%, 3.9 ± 2.3%, and 0% of the values for untreated cells after 1, 3, 7, and 9 days incubation with ATc, respectively (Fig. 2D)Citation . In similar experiments, ATc-mediated down-regulation of HER-2/neu was examined by immunohistochemistry. The number of HER-2-positive tumor cells was determined in three independent experiments and expressed as a percentage of all tumor cells (Fig. 2E)Citation . Mean values (±SD) were 83.9 ± 11.3%, 35.8 ± 6.0%, 11.5 ± 11.2%, and 3.97 ± 3.97% of the values for untreated cells after 1, 3, 7, and 9 days incubation with ATc, respectively (Fig. 2E)Citation . Similarly, flow-activated cell-sorting analysis showed a clear down-regulation of HER-2 during incubation with ATc (Fig. 2F)Citation . Antibodies against the extracellular (Ab-5) and the intracellular (Ab-3) domains of HER-2 were used. For both the extra- and intracellular domains, clear down-regulation was observed during incubation with ATc. Interestingly, the HER-2/neu down-regulation was more pronounced for the extracellular compared with the intracellular domain, whereas an antibody directed against the extracellular domain of rat HER-2 (Ab-4), serving as a negative control, was not affected by ATc (Fig. 2F)Citation .



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  		Fig. 2. Down-regulation of HER-2/neu in NIH3T3-her2 cells by ATc in vitro. A, Western blot analysis after 1, 3, and 7 days of incubation with 10 ng/ml ATc. B, loading control with anti-ß-actin staining. C, Coomassie-stained membrane. D, densitometric analysis of Western blots. Mean (SD; bars) values of three independent experiments are shown. *, below detection limit. E, immunohistochemical analysis after 1, 3, 7, and 9 days of incubation with 10 ng/ml ATc showing percentage of HER-2-positive cells. Mean (SD; bars) values of four independent experiments are shown. F, flow-activated cell-sorting analysis of HER-2 after 4, 12, and 24 h and 3 and 7 days of incubation with 10 ng/ml ATc. Antibodies directed against the extracellular domain (Ab-5; {blacksquare}) and the intracellular domain (Ab-3; ) of HER-2 were used. An antibody specific for the extracellular domain of rat HER-2 (Ab-4; ) was used as a negative control. Shown is one representative set of data of three independent experiments.

 
Conditional Expression of HER-2/neu in Vivo.
NIH3T3-her2 cells were injected s.c. into nude mice. After 16–20 days s.c.-growing tumors reached a volume of 0.9–1.4 cm3. Mice were then treated with daily s.c. injections of 10 mg ATc/kg of body weight for 1, 3, and 7 days. Western blot analysis showed a marked decrease in HER-2 expression as early as 24 h after the first injection of ATc (Fig. 3A)Citation . After 3 days, HER-2 was no longer detectable by Western blotting. Mean (±SD) values after densitometric analysis were 18.2 ± 6.9% after 1 day and 0% after 3 and 7 days under administration of ATc compared with time-matched control tumors (Fig. 3D)Citation . Thus the decrease in HER-2 expression after exposure to ATc was even faster in vivo than in the in vitro experiments. Mice not receiving ATc were used as negative controls. When ATc-treated tumors were harvested after 1, 3, and 7 days, time-matched control tumors were harvested simultaneously. Thus, the three controls shown in Fig. 3ACitation are of incremental ages matching those of the tumors treated with ATc for 1, 3, and 7 days. Obviously, HER-2 expression increased with tumor growth (Fig. 3ACitation , Lanes 1–3). In similar experiments, the ATc-mediated decrease in HER-2 expression was examined by immunohistochemistry. A clear decrease in HER-2 expression was observed during administration of ATc. After 7 days, HER-2 was no longer detectable. Mean (±SD) values for the HER-2-positive cells were 62.5 ± 3.6%, 2.8 ± 2.3%, and 0% on days 1, 3, and 7 of ATc administration, respectively (Fig. 3E)Citation .



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  		Fig. 3. Down-regulation of HER-2/neu in NIH3T3-her2 tumors by anhydrotetracycline (ATc) in vivo. A, Western blot analysis of human HER-2 in s.c. tumors of nude mice induced after s.c. injection of NIH3T3-her2 cells. Decreases in HER-2 expression were observed 1, 3, and 7 days after administration of ATc to nude mice. The controls without ATc treatment were included. The age of the three controls corresponded to the age of the treated tumors (1, 3, and 7 days), respectively. B, loading control with anti-ß-actin staining. C, Coomassie-stained membrane. D, mean (SD; bars) values of three mice from three independent studies are shown for each time point. E, immunohistochemical analysis of human HER-2 in s.c. tumors in nude mice at 0, 1, 3, and 7 days of therapy with daily doses of 10 mg ATc/kg of body weight. Mean (SD; bars) values from one representative study are shown. Two independent experiments were performed. *, below detection limit.

 
Down-Regulation of HER-2/neu Leads to Tumor Remission.
To develop a treatment model, nude mice were injected s.c. with 7 x 106 NIH3T3-her2 cells. Eight to 10 days after injection, small tumors became visible (Fig. 4)Citation . Tumors were allowed to reach different volumes (Table 1)Citation . Mice were then randomized into ATc-treated and control groups. The ATc-treated group received daily s.c. injections of 10 mg ATc/kg of body weight. The day of the first administration of ATc was defined as day 0. Representative examples of one ATc-treated mouse from group I and one control mouse are shown in Fig. 4ACitation . Tumor remission under ATc treatment (Fig. 4BCitation , IV–VI) as well as further tumor growth in the control mouse (Fig. 4BCitation , VII–IX) were also visualized by MRI using gadolinium–diethylenetriaminepentaacetic acid as a contrast agent. In groups I and II, mice with mean tumor volumes of 1.2 and 3.9 cm3 were treated with 7 subsequent daily doses of ATc. Strong remission was observed for both groups (Fig. 4, C and D)Citation . In contrast, a continuous increase was seen for control tumors. A longer follow-up of groups I and II was not possible because the tumors were subsequently isolated for analysis of HER-2 expression, proliferation, and apoptosis.



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  		Fig. 4. Tumor remission after down-regulation of HER-2. A, tumor growth in two representative nude mice. I—III show an ATc-treated mouse (daily s.c. injections of 10 mg ATc/kg) on the left and a control mouse on the right. B, the same tumors were also visualized by MRI. IV–VI show tumor remission during ATc administration, whereas VII–IX show further tumor growth in the control mouse. Pictures were taken on days 0 (I, IV, and VII), 4 (II, V, and VIII), and 6 (III, VI, and IX) of ATc administration. The day of the first administration of ATc was defined as day 0. C, ATc treatment of tumors with a mean volume of 1.2 cm3 (group I). Tumors were induced by s.c. injection of 7 x 106 NIH3T3-her2 cells in nude mice. One group of mice ({blacksquare}; n = 6) received seven daily injections of 10 mg ATc/kg of body weight as soon as the mean tumor volume reached 1.2 cm3. The second group of mice ({square}; n = 6) served as controls. Data are mean (SD; bars) values of six mice. D, ATc treatment of tumors with a volume of 3.9 cm3 (group II). Experiments were performed similarly to those described in the legend for C. E, ATc treatment of tumors with a volume of 0.2 cm3 (note the different scale of the Y axis). Experiments were performed as described in the legend for C, with the exception that 37 subsequent daily doses of ATc were given. F, ATc treatment of tumors with a volume of 0.8 cm3. Experiments were performed as described in the legend for C, with the exception that 40 subsequent daily doses of ATc were given. G, ATc treatment of tumors with a volume of 1.9 cm3. Experiments were performed as described in legend for E. H, ATc treatment of tumors with a volume of 14.9 cm3 (note the different scale of the Y axis). Thirty subsequent daily doses were given. The Western blot (inset) was performed with tumor tissue obtained at day 30. Lane 1, tissue from one of these recurrent tumors (negative); Lane 2, NIH3T3-her2 cells as positive controls; Lanes 3 and 6, molecular weight marker; Lanes 4 and 5, loading control for Lanes 1 and 2 using anti-ß-actin staining.

 
Tumor Remission Depends on Initial Tumor Volume.
To examine whether the tumor volume at the onset of ATc administration was relevant for tumor remission, we examined four groups of mice with tumors 0.2, 0.8, 1.9, and 14.9 cm3 in volume. Mice were treated with up to 40 subsequent daily doses of 10 mg/kg ATc (Fig. 4, E–H)Citation . The results show that the extent and duration of tumor remission depended on the initial tumor volume. The 0.2-, 0.8-, 1.9-, and 14.9-cm3 tumors showed 100, 98.1, 81.4, and 74.2% reductions in tumor size, respectively, after 7 days of ATc administration. Clear differences among the four groups were obtained by the Kruskal–Wallis test (P = 0.005; Fig. 4, E–HCitation ).

To determine whether the chance to achieve complete remissions depended on initial tumor volume, we gave daily injections of ATc to additional mice with tumors 0.2 cm3 (n = 7), 0.8–1.2 cm3 (n = 10), and >=1.9 cm3 (n = 11) in volume (Table 2)Citation . Complete remissions were achieved in 100, 40, and 18% of the mice with small- (0.2 cm3), intermediate- (0.8–1.2 cm3), and large-sized (>=1.9 cm3) tumors (P = 0.003, {chi}2 test; Table 2Citation ). Thus, tumor remission clearly depended on initial tumor size at the onset of ATc administration.


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  		Table 2 Number of complete remissions of 0.2, 0.8–1.2, and >=1.9 cm3 NIH3T3-her2 tumors in nude mice

The chance for complete remissions of NIH3T3-her2 tumors in nude mice after switching off of HER-2 expression depended on the tumor volume at the onset of ATc administration. A clear difference in the frequency of complete remissions was observed among the three groups of mice with different tumor volumes (P = 0.003, {chi}2 test).

 
Recurrent tumor growth was observed in all mice, even in those with previous complete remissions. The time period for which mean tumor volume could be suppressed to volumes <=0.1 cm3 under ATc administration was 34, 22, 8, and 0 days for tumors with an initial tumor volume of 0.2, 0.8, 1.9, and 14.9 cm3, respectively (Fig. 4, E–H)Citation . Thus, clear differences depending on initial tumor volumes were obtained (P = 0.005, Kruskal-Wallis test). Thereafter, tumors continued to grow. The recurrent tumors were analyzed for HER-2/neu expression. In all seven recurrent tumors analyzed, HER-2 expression was below detection limit (Figs. 4HCitation and 5ACitation ). To demonstrate the magnitude of the change in HER-2 expression between primary and recurrent tumors, we performed a limited dilution series. Proteins from a primary tumor that had not been exposed to ATc were diluted in six dilution steps by a factor of 5. A dilution of 54 still showed a clear signal for HER-2 (Fig. 5C)Citation . Thus, HER-2 is at least 625-fold down-regulated in recurrent tumors compared with primary tumors.



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  		Fig. 5. HER-2/neu expression in recurrent tumors. A, Western blot analysis of six recurrent NIH3T2-her2 tumors in nude mice that were still receiving ATc. An untreated NIH3T3-her2 tumor (Co.) and tumors treated with ATc for 1, 3, and 7 days served as controls. B, loading control with anti ß-actin staining. C, limited dilution of protein obtained from a NIH3T3-her2 control tumor in nude mice to demonstrate the detection limit of HER-2 Western blot analysis. Protein extract from a NIH3T3-her2 control tumor was diluted 6 times by a factor of 5 and compared with two recurrent tumors for HER-2 expression. D, loading control for the limited dilution series and the two recurrent tumors with anti-ß-actin staining.

 
Injection of NIH3T3 wild-type cells and NIH3T3 cells transfected with the empty vector (pTBC/SEAP without HER-2; Ref. 21 ), pTBC1 Hygro R (21) , and pUHD 15-1 (22) caused no tumor growth the first 25 days. Only after longer periods were small, very slowly proliferating tumors observed (data not shown).

Inhibition of Proliferation after HER-2/neu Down-Regulation.
The influence of HER-2/neu down-regulation on proliferation was determined by Ki-67 immunohistochemistry. For this purpose, exponentially growing NIH3T3-her2 cells were incubated with 10 ng/ml ATc for 0 (controls), 1, 3, and 7 days. Before the addition of ATc, ~84% of the nuclei were Ki-67 positive (Fig. 6, A and B)Citation . During 7 days of incubation with ATc, the number of Ki-67-positive nuclei decreased to a mean of 23.5% (Fig. 6B)Citation . Similar results were obtained in vivo. Tumors in nude mice showed a marked decrease in Ki-67-positive nuclei (Fig. 6C)Citation . The frequency of Ki-67-positive cells increased from 30.3 to 41% in untreated tumors during the experimental period of 7 days (Fig. 6D)Citation . In contrast, ATc-treated tumors showed only 10.3 and 8.3% Ki-67-positive nuclei after 3 and 7 days of ATc treatment, respectively (Fig. 6D)Citation .



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  		Fig. 6. Proliferation is influenced by down-regulation of HER-2/neu. A, immunohistochemical analysis of Ki-67 in NIH3T3-her2 cells in vitro after 1, 3, and 7 days of incubation with 10 ng/ml ATc. B, determination of the percentage of Ki-67-positive cells in vitro. Data are mean (SD; bars) values and ranges for two independent experiments. C, Ki-67 immunohistochemistry for ATc-treated tumors in nude mice and time-matched controls. D, mean (SD; bars) values from one representative immunohistochemical study are shown. Two independent experiments were performed.

 
Induction of Apoptosis as a Consequence of HER-2/neu Down-Regulation.
The number of TUNEL-positive NIH3T3-her2 cells increased in vitro, amounting to 2.2 ± 2.5% positive nuclei in control cells and 17.5 ± 12.1%, 29.3 ± 15.2%, and 28.8 ± 11.8% positive nuclei after 1, 3, and 7 days of incubation with ATc, respectively (Fig. 7, A and B)Citation . In s.c.-growing tumors in nude mice, an increase in TUNEL positive cells could also be seen on days 1, 3, and 7 of administration of ATc (Fig. 7, C and D)Citation . In vivo, the variation in the number of TUNEL-positive cells was relatively large in the time-matched controls. However, at days 1, 3, and 7 of ATc administration, the ATc-treated tumors showed higher percentages of TUNEL-positive cells compared with the time-matched controls. On day 7, 62.5% of the ATc-exposed tumor cells were TUNEL positive (Fig. 7D)Citation , which was 2-fold higher than in NIH3T3-her2 cells incubated with ATc for 7 days in vitro (Fig. 7B)Citation .



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  		Fig. 7. Down-regulation of HER-2/neu induces apoptosis. A, apoptosis as evidenced by the TUNEL assay in NIH3T3-her2 cells in vitro during incubation with 10 ng/ml ATc. Representative examples of NIH3T3-her2 cells after 1, 3, and 7 days of incubation with ATc. B, mean (SD; bars) values for four independent in vitro experiments are shown. C, apoptosis as evidenced by the TUNEL assay in tumors in nude mice under therapy with ATc. Representative examples of a control tumor and tumors after 1, 3, and 7 days of therapy with ATc are shown. D, mean (SD; bars) values from one representative in vivo study are shown. Two independent experiments were performed. E, apoptosis as evidenced by Western blot analysis of caspase-3 in tumors in nude mice under therapy with ATc. The anti-caspase-3 antibody detects the inactive zymogen (32–35 kDa) and the active cleaved large fragment of caspase-3 (17–20 kDa). The Western blot shown is a representative of three independent experiments. F, loading control with anti-ß-actin staining. G, Coomassie-stained membrane.

 
To confirm the positive result of the TUNEL assay in vivo, we also visualized the cleavage of inactive caspase-3 by Western blot analysis (Fig. 7E)Citation . In s.c.-growing NIH3T3-her2 tumors in nude mice, inactive caspase-3 was decreased after 3 and 7 days of ATc treatment. In contrast, the active form of caspase-3 was increased after 1 day of treatment with ATc (Fig. 7E)Citation . After 3 days of ATc treatment, the active form of caspase-3 decreased again and was no longer detectable after 7 days of treatment. It should be noted that after 3 and 7 days, the tumor cells were deteriorating, which can be seen from the strong tumor remission in panels C–H in Fig. 4Citation . Similar to the TUNEL assay, in which relatively high numbers of positive cells were seen in control tumors (Fig. 7D)Citation , a relatively high amount of active caspase-3 was detectable in untreated tumors (Fig. 7E)Citation . However, both, the TUNEL and caspase-3 assays showed pronounced induction of tumor cell apoptosis after ATc-mediated down-regulation of HER-2/neu.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Immunohistochemistry
 Flow Cytometry
 Statistical Analysis
 RESULTS
 DISCUSSION
REFERENCES
 
Overexpression of the HER-2 oncogene in mammary tissue of transgenic mice leads to breast cancer (6) . HER-2/neu amplification and/or overexpression in human breast and ovarian cancer is associated with tumor progression and adverse prognosis (12) . Trastuzumab (Herceptin), a humanized monoclonal antibody directed against HER-2, provides clinical benefits for patients diagnosed with advanced breast cancer that overexpress the HER-2 protein (27 , 28) . Several mechanisms contribute to the beneficial effects of trastuzumab. Trastuzumab acts as an antiangiogenic substance that leads to regression of the vasculature in experimental human breast tumors in mice (29) . Several lines of evidence suggest that HER-2 down-regulation might induce apoptosis in tumor cells (16 , 30, 31, 32, 33, 34, 35, 36) . In these studies, down-regulation of HER-2 was achieved by Herceptin (16) , adenoviral type 5 E1A (32 , 35 , 36) , phosphorothioate antisense oligodeoxyribonucleotides (34) , hammerhead ribozymes (31) , or by pharmacological antagonists of the HER-2 signal transduction pathway (30 , 33) . However, the effect of HER-2/neu expression on apoptosis is controversial because some investigators reported that, in contrast to the above reports, HER-2/neu overexpression is associated with enhanced apoptosis (37, 38, 39) .

To investigate the cellular functions of HER-2/neu, we developed in vitro and in vivo models of conditional human HER-2/neu expression that used the TET-OFF system, based on a tetracycline-controlled transactivator protein, tTA (21 , 22) . In vitro, expression of HER-2 could be down-regulated below the detection limit by incubation of tumor cells with ATc. To examine the influence of HER-2 in vivo, we injected NIH3T3-her2 cells s.c. into nude mice. Small tumors were already visible after 8–10 days and continuously increased in size, reaching mean diameters of 3.0 cm ~30 days after injection. In contrast, wild-type NIH3T3 cells and NIH3T3 cells injected with the empty vector did not form tumors up to day 25.

Tumors induced after subcutaneous injection of NIH3T3-her2 cells were reversible by treatment of mice with ATc. To examine whether tumor remission depends on the initial tumor volume before down-regulation of ATc, we treated four groups of mice with very early (0.2 cm3), early (0.8 cm3), intermediate (1.9 cm3), and advanced (14.9 cm3) tumors with ATc for up to 40 consecutive days. Our results indicated that tumor remission does depend on the initial tumor volume. Seven days after the onset of HER-2 down-regulation, tumor size was reduced by 100, 98.1, 81.4, and 74.2% for tumors with initial volumes of 0.2, 0.8, 1.9, and 14.9 cm3, respectively. The time periods for which mean tumor volume could be suppressed to volumes <0.1 cm3 under ATc administration were 34, 22, 8, and 0 days in the same groups of mice. The probability of achieving complete remissions also depended on initial tumor volume at the onset of ATc administration: complete remission could be reached as a consequence of HER-2 down-regulation in 100, 40, and 18% of mice with small (0.2 cm3), intermediate (0.8–1.2 cm3), and large (>1.9 cm3) tumors, respectively. However, for all tumors, even for those that apparently underwent complete remission, recurrent tumor growth occurred within 45 days despite ATc administration. Western blot analysis demonstrated that growth of all recurrent tumors was HER-2 independent. Limited dilution experiments showed that HER-2 is at least 625-fold down-regulated in the recurrent, HER-2-independent tumors compared with primary tumors. We speculate that additional genetic events, such as new mutations, must have occurred, causing HER-2-independent tumor growth. It will be of high importance to identify these new mutations (second hits) because this may lead to new strategies for improving HER-2/neu-blocking tumor therapy. Obviously, for advanced tumors the probability of additional hits causing HER-2-independent growth is higher compared with small tumors.

Our results show that the efficacy of HER-2-blocking therapy strategies depends on tumor size. To our knowledge no clinical study has been performed to date for Herceptin efficacy in different stages of disease. However, our data may encourage the development of HER-2-targeted therapeutic strategies as first-line therapies for small tumors rather than the limiting of this treatment modality to advanced node-positive disease. The clinical implication of this observation seems obvious; Herceptin can be expected to be most efficient for treatment of tumors with low volumes and low staging. For tumors with a relatively high residual tumor volume after surgery, e.g., many node-positive breast carcinomas or recurrent tumors, the effect of Herceptin can be expected to cause a temporary reduction in tumor volume but no long-term remission.

The rapid remission of tumors after switching off of HER-2 expression was more than we had expected, considering the relatively large sizes of tumors at the beginning of ATc administration (0.2, 0.8, 1.9, 1.2, 3.9, or 14.9 cm3). We therefore examined the mechanisms responsible for tumor remission. During 7 days of ATc administration, the percentage of Ki-67-positive cells decreased from ~40% to 8.3%. Although such a block in cell cycle progression could explain the lack of additional tumor growth, it cannot account for the observed tumor remission. We therefore analyzed apoptosis by two independent assays, the TUNEL assay and Western blot analysis, to detect the inactive and the active forms of caspase-3. Both assays demonstrated an induction of apoptosis as a consequence of ATc-mediated down-regulation of HER-2. The TUNEL assay showed an increase in TUNEL-positive cells up to day 3 of ATc administration, whereas the inactive form of caspase-3 decreased up to day 7. A clear increase in the active form of caspase-3 was seen as early as 24 h after initiation of ATc treatment. However, after 3 and 7 days of ATc administration, the active form of caspase-3 decreased dramatically. Most probably, this can be explained by further degradation of active caspase-3. This seems plausible because a strong remission of tumor volume occurred after 3 and 7 days of ATc administration. In addition, histological analysis of tumors treated for 3 and 7 days with ATc showed a strong decrease in viable cells with intact nuclei and a strong increase in dead tumor mass (Fig. 6C)Citation .

Our data suggest that antagonization of HER-2 may not only stop proliferation but also induce apoptosis and tumor remission. However, it must be kept in mind that this scenario may be observed only under highly specific experimental circumstances. In human tumors, several oncogenes and tumor suppressor genes are dysregulated in concert (40, 41, 42) . In contrast, in the present tumor model, the tumorigenic property of NIH3T3-her2 cell-derived tumors is based on overexpression of a single oncogene, her-2. Interestingly, tumors in our NIH3T3-her2 model were reversible, possibly because only a single oncogene was dysregulated. Reversibility was rapidly lost in large NIH3T3-her2 tumors with a mean volume of 14.9 cm3 as well as in some smaller tumors, but after longer latency periods. Probably, activation of other oncogenes or inactivation of tumor suppressor genes led to HER-2-independent tumor growth. We are aware of the fact that our model is artificial. Nevertheless, it offers the opportunity to study the direct consequences and mechanisms attributable to a single oncogene.

At present, no experimentally proven molecular mechanism can explain why down-regulation of HER-2 not only stops further proliferation but also leads to induction of apoptosis and tumor remission. One can speculate that the extreme overexpression of HER-2 may constitute a strong antiapoptotic signal, mediated by AKT kinase or by nuclear factor-{kappa}B (Fig. 1ACitation ; Refs. 8 , 43 ). In response to the antiapoptotic signaling, cells might up-regulate some pro-apoptotic pathways that will be without major consequences attributable to the strong antiapoptotic signaling. When the antiapoptotic signaling mediated by HER-2 is suddenly switched off by ATc, the sensitive balance between cell survival and cell death may be disturbed, and the compensatory up-regulated proapoptotic mechanisms, which probably cannot be down-regulated fast enough, will dominate and lead to cell death.

Our results show once again that overexpressed oncogenes represent an attractive therapeutic target and that their down-regulation can disturb the delicate balance between pro- and antiapoptotic mechanisms and may be of therapeutic benefit. Tumor remission as a consequence of oncogene down-regulation has previously been shown for mutated H-RAS(44) . In that study, a TET-regulated H-RASV12G transgenic mouse carrying valine instead of glycine in position 12 of H-RASwas generated. The TET-ON system was used (22) for expressing the H-RASV12G transgene only in the presence of doxycycline. Doxycycline-induced expression of H-RASV12G led to nonmetastatic melanomas in this transgenic mouse model. However, INK4a-deficient mice had to be used to achieve development of melanomas (44) . Withdrawal of doxycycline and the resulting H-RASV12G down-regulation led to regression of melanomas. In this aspect, the results reported by Wong and Chin (44) are similar to those of our study, in which ATc-induced down-regulation of HER-2, an upstream member of the RAS pathway, led to remission of tumors.

After submission of this manuscript, an article was published (45) that presents data on mice conditionally overexpressing HER-2/neu in the mammary gland. Similar to our study, Moody et al. (45) reported that tumors conditionally overexpressing HER-2/neu depend on continued HER-2 expression for maintenance. A new finding in our study is the relationship between tumor size and HER-2 independence.

In conclusion, we have shown that switching off of human HER-2 expression by the TET-OFF system in s.c. tumors in mice leads not only to a halt in proliferation but also to apoptosis in tumor cells and tumor-size-dependent remission.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by the Deutsche Forschungsgemeinschaft (Project HE2509) and the Stiftung Rheinland-Pfalz for Innovation. Back

2 To whom requests for reprints should be addressed, at Institute of Toxicology, Johannes Gutenberg-University, Obere Zahlbacherstrasse 67, 55131 Mainz, Germany. Phone: 49 6131 39-30028; Fax: 49 6131 230506; E-mail: schiffer{at}mail.uni-mainz.de Back

3 The abbreviations used are: MAP, mitogen-activated protein; ATc, anhydrotetracycline hydrochloride; MRI, magnetic resonance imaging; TBS, Tris-buffered saline; TUNEL, terminal dUTP deoxynucleotidyltransferase nick end labeling. Back

Received 9/27/02. Revised 8/ 6/03. Accepted 9/ 5/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Immunohistochemistry
 Flow Cytometry
 Statistical Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 

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