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[Cancer Research 60, 3218-3224, June 15, 2000]
© 2000 American Association for Cancer Research

Experimental Therapeutics

Cancer Gene Therapy by Thyroid Hormone-mediated Expression of Toxin Genes1

Verónica Martín, Maria Luisa Cortés, Pablo de Felipe, Antonella Farsetti, Nora B. Calcaterra and Marta Izquierdo2

Departamento de Bioquímica y Biología Molecular, Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Facultad de Ciencias, Cantoblanco 28049 Madrid, Spain [V. M., M. L. C., P. d. F., M. I.]; Institute Medicina Sperimentale, Consiglio Nazionale delle Ricerche and Laboratorio di Oncogenesi Molecolare, Institute Regina Elena, 00161 Rome, Italy [A. F.]; and Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nac. Rosario, 2000-Rosario, Argentina [N. B. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 Inoculation of Tumor Cells
 Treatment of Induced C6...
 MRI
 RESULTS
 DISCUSSION
 REFERENCES
 
Many of the strategies developed in the last few years to treat cancer by gene therapy are based on putative killer-suicide genes whose products convert a prodrug into a toxic compound. When the therapy is applied to humans, a vector carrying the killer gene is first inoculated into the tumor of the patient, who 1 week later receives the corresponding prodrug that will selectively kill the cells able to process it to its toxic derivative. A strategy that obviates the need for a prodrug to destroy the cancer cells would be preferable because the patient would only need one treatment instead of two consecutive ones. In the following study, we describe the construction of retroviral vectors in which a reporter or a toxin gene (either the Pseudomonas exotoxin or the Ricinus communis toxin, ricin) is placed under the control of the thyroid hormone (T3) regulatable promoter of the rat myelin basic protein (MBPp). We demonstrate that the expression of these genes under the control of MBPp is regulated by T3 in vitro and in vivo. In vitro, the MBPp is switched off when T3 is removed from the serum of the culture medium, allowing the production of retroviruses carrying the toxic gene. In vivo, the toxin gene bearing retroviruses is capable of eradicating experimentally induced brain tumors in Wistar rats. The gene therapy strategy described here does not require the use of a prodrug to destroy the neoplastic cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 Inoculation of Tumor Cells
 Treatment of Induced C6...
 MRI
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite their regional pattern of growth and progression, malignant gliomas are the third leading cause of death from cancer in persons 15–34 years of age. This has led to the development of a series of gene therapy protocols based mainly on the herpes simplex virus thymidine kinase/ganciclovir system (1, 2, 3, 4, 5, 6, 7, 8, 9) . However, total remission of human malignant brain tumors has not been achieved. The grim prognosis for patients with gliomas is related to a lack of potent agents with adequate tumor specificity.

Over the last decade, a significant number of tumor tissue-selective promoters and enhancer elements have been isolated that have the potential to be used in gene therapy of malignancies (10) . The regulation of specific genes is usually accomplished through the binding of transcription factors and associated proteins. The MBP,3 the second most abundant protein in the CNS (11) , contains an upstream regulatory sequence that confers cell type- and stage-specific transcription to MBP expression in oligodendrocytes during brain development (12, 13, 14) . Transcriptional regulation analyses of the mbp gene in vitro do not show as clear a cell specificity as that seen in vivo (15, 16, 17, 18, 19) . Nevertheless, a T3 response element has been characterized within the MBPp. The hormone-receptor complex binds to this thyroid hormone response element to activate transcription, whereas repression is observed in the absence of the hormone (20) .

We engineered a series of retroviral vectors to test the applicability of a MBPp/T3 regulatable gene therapy system that would allow the expression of a toxic gene in the tumor cells while being able to tightly repress it in retroviral producer cells ({Psi}CRIP). Vectors carrying the luc reporter gene under the control of the MBPp showed high luc activity in the presence of T3 and showed virtually no activity in its absence. By replacing the reporter gene with a toxin gene, we obtained toxic retrovirus producer cells only in the absence of T3. The presence of T3 in vitro or in vivo (endogenous T3) allowed a partial or total growth arrest of infected cell lines. Finally, we showed a total remission of induced brain tumors in Wistar rats treated with toxin-retrovirus producer cells. These results indicate the feasibility of using a regulatable retroviral toxin gene therapy system for glioblastoma treatment.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
Inoculation of Tumor Cells
 Treatment of Induced C6...
 MRI
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Animal Model
Ecotropic retroviral packaging {Psi}CRE cells and amphotropic retroviral packaging {Psi}CRIP cells were grown in DMEM supplemented with 10% heat-inactivated calf serum. Rat glioblastoma C6 cells and human glioblastoma U-373-MG cultures were also grown in DMEM but were supplemented with 10% heat-inactivated FCS. All cells were kept at 37°C in a humidified incubator with 7% CO2 and 97% relative humidity. Sera were depleted of T3 using a HCl-Tris equilibrated ionic exchange AG-1X8 (Bio-Rad) resin (21) .

The animals used were 2- or 3-month-old BALB/c females, SCID mice, and Wistar rats.

Construction of Retroviral Vectors
The luc gene was amplified by PCR using primers 5'-luc-BAMHI-GTGTTGGATCCATGGAAGACGCCAAAAAC and 3'-luc-BAMHI-CAGTGGATCCTTACAATTTGGACTTTCC and cloned into the BamHI site of the Y plasmid (Ref. 22 ; pBabePuro-derived plasmid; Fig. 1Citation ) to generate the plasmid retro-luc. The 13-MBPp and 256-MBPp were obtained by HindIII/XhoI restriction enzyme digestion from the MBP1317CAT and MBP256CAT plasmids, respectively (17) , and cloned into retro-luc (SalI site), creating plasmids retro-1.3MBPp-luc and retro-256MBPp-luc. To obtain retro-1.3MBPp-pe-toxin and retro-1.3MBPp-ri-toxin plasmids, the luc gene was removed by BamHI restriction enzyme treatment and substituted with either the HindIII/XhoI fragment corresponding to the ricin gene from pEMBLyex4RA (23) or the XbaI-EcoRI cut pe gene (24) obtained from plasmid pVC45F (kindly provided by I. Pastan, National Cancer Institute, NIH, Bethesda, MD). The pRSVTRß1 plasmid used in cotransfection experiments contains the rat T3 receptor gene (25) downstream from the RSV promoter (17) . The LNCXTRß1 plasmid contains the HindIII-HpaI TRß1 fragment from pRSVTRß1 under the control of the CMV promoter.



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  		Fig. 1. Structure of the different retroviral vectors constructed. The packaging sequence from wild-type Moloney leukemia retrovirus is represented as {Psi}; arrows represent gene transcription orientation. A, Y vector carrying only the pac gene under the control of the 5'-LTR promoter. B, retro-luc vector carrying the luc gene under the control of the 5'-LTR promoter and the SV40 promoter directing pac gene expression. C, retro-1.3 MBPp-luc vector carrying the pac gene under the control of the 5'-LTR; luc expression is under the control of the 1300-bp MBPp, both of which were placed in the opposite orientation to pac gene; retro-1.3MBPp-pe-toxin and retro-1.3MBPp-ri-toxin are constructed by replacing the luc gene of retro-1.3MBPp-luc with the pe gene or A chain of the ricin gene, respectively. D, retro-256MBPp-luc is the same as retro-1.3MBPp-luc (B) retroviral vector; the only difference is the length of the cloned MBPp. The retro-256MBPp-luc vector has only the first 256 bp of the large MBPp. E, LNCXTRß1 is a vector that has the TRß1 under the control of the CMV promoter and the neomycin resistance gene under the control of the left LTR. Both genes are cloned in the same orientation (5'-3').

 
Transfection and Infection
Plasmids were transfected using either Lipofectin (Life Technologies, Inc.) or Tfx50 (Promega), following the protocols provided by the manufacturers. Producer cells were obtained by transfection of ecotropic packaging {Psi}CRE cells and subsequent harvesting of the supernatant carrying the viral particles to infect the amphotropic packaging cell line {Psi}CRIP, according to a method described previously (26) . The supernatants of the transduced {Psi}CRIP cells were used to infect the C6 and U-373-MG cell lines. Transfected and infected cells were selected in 2 µg/ml puromycin, and the colonies obtained were pooled or cloned. Titers were estimated by infecting NIH-3T3 cells as described previously (4) .

Luc Activity Assays
Cells (104) growing as monolayers were washed once with PBS, resuspended in 100 µl of extraction buffer [100 mM potassium phosphate (pH 7.8), 1 mM DTT, and 0.5% Triton X-100], and incubated at 4°C for 5 min. Extracts were clarified by centrifugation at 300 x g, and supernatants were collected for analysis. Assays were performed using 20 µl of each extract plus 100 µl of reaction buffer [17.5 mM potassium phosphate (pH 7.8), 13 mM MgCl2, 12.5 mM ATP (pH 7.77), 52.5 µM DTT, and 134 µg/ml BSA] and 100 µl of 1 mM luciferin. The number of relative light units produced during the first 10 s was recorded using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA).

Immunofluorescence Assay
After 48 h in the presence or absence of T3, the cells were washed with PBS and fixed in methanol at -20°C for 5 min. The cells were then blocked in PBS plus 1% BSA. This buffer was used in subsequent washing and antibody incubation steps. Cells were washed and incubated for 1 h at 37°C with a rabbit anti-pe A antibody (Sigma) and incubated for a second hour with an antirabbit fluorescein or Texas red-conjugated secondary antibody (Amersham). After hybridization and the subsequent washing steps, the cells were mounted with Moviol 4-88 and the antibleaching reagent diazabicyclo[z.z.z]octane (DABCO). Microscopy was performed on a Zeiss Axiovert microscope.

Cell Death Analysis
Measurement of [3H]Thymidine Incorporation into DNA.
Cells (2 x 104) growing as monolayers were labeled for 6 h with 8 µCi of [3H]thymidine (Amersham) per well, trypsinized, and precipitated with 2 ml of 10% trichloroacetic acid. The incorporated radioactivity was measured by liquid scintillation counting. The results are the average of three independent experiments in which three identical sample points were measured each time.

Methylene Blue Staining.
Cells were fixed in 12% glutaraldehyde, stained with 0.05% methylene blue, and eluted in 0.33 N HCl. Absorbance at 630 nm was determined in a MR 5000 microplate reader (Dynatech, West Sussex, United Kingdom). The results are the average of two independent experiments in which six identical samples were measured.

[35S]Methionine/[35S]Cysteine Protein Incorporation.
Infected cells (104) were cultured in the absence or presence of T3. The cells were pulse-labeled for 1 h with a mixture of [35S]methionine and [35S]cysteine (150 µCi/106 cells; Amersham). Lysates were precipitated in 10% trichloroacetic acid, and the label was measured in a scintillation counter.


   Inoculation of Tumor Cells
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Inoculation of Tumor Cells
Treatment of Induced C6...
 MRI
 RESULTS
 DISCUSSION
 REFERENCES
 
s.c. Injection.
To investigate tumor growth induction in vivo, we injected 5 x 105 retro-1.3MBPp-luc-infected C6 cells s.c. into the left flank of six SCID mice. Three of these mice received injections of 5 x 105 retro-1.3MBPp-ri-toxin-infected C6 cells in the right flank, and the other three mice received injections of 5 x 105 retro-1.3MBPp-pe-toxin-infected C6 cells in the right flank. When tumors on the left side became palpable 10 days later, the mice were killed, and all tumors were removed and weighed.

Twenty BALB/c mice had 5 x 105 retro-1.3MBPp-luc-infected C6 cells injected in the left flank. Ten of these mice had 5 x 105 retro-1.3MBPp-ri-toxin-infected C6 cells injected in the opposite flank, and the other 10 mice had 5 x 105 retro-1.3MBPp-pe-toxin-infected C6 cells injected in the right flank. After 15 days, the mice were killed, and the presence or absence of tumor was determined. Injections were performed in a similar way in the right flank of 10 Wistar rats: 5 rats were injected with 5 x 105 retro-1.3MBPp-ri-toxin-infected C6 cells; and the other 5 rats were injected with 5 x 105 retro-1.3MBPp-pe-toxin-infected C6 cells. All received injections of 5 x 105 retro-1.3MBPp-luc-infected C6 cells in the left flank.

Intratumoral Injection.
We have essentially followed the procedures described previously (3 , 4) . Briefly, Wistar male rats weighing 250–300 g were anesthetized with an inhalation mixture of 0.8 liter/min oxygen (O2), 0.4 liter/min protoxide (N2O), and 3% isofluorane gas (Forane) before placing them in a stereotactic apparatus. During the operation, the same anesthetic was maintained. Five x 105 retro-1.3MBPp-luc-, retro-1.3MBPp-ri-toxin-, or retro-1.3MBPp-pe-toxin-infected C6 cells or 5 x 106 retro-1.3MBPp-pe-toxin-retrovirus-producing {Psi}CRIP cells were injected at a concentration of 105 cells/µl in complete PBS (containing calcium and magnesium) supplemented with 0.1% glucose. With the aid of the manipulating arm of the stereotactic apparatus, a total of 5 µl was introduced over a 5-min interval into the frontoparietal lobe of the right cerebral hemisphere (4 mm to the right from the bregma and 4.5 mm deep from the skull) using a 5-µl Hamilton syringe connected to a 26-gauge needle; the needle was kept in place 3 min before and after injection. Two weeks later, magnetic resonance visualization of the tumor and size estimation were performed.


   Treatment of Induced C6 Wistar Rat Brain Tumors with {Psi}CRIP-retro-1.3MBPp-pe-toxin Producer Cells
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Inoculation of Tumor Cells
 Treatment of Induced C6...
MRI
 RESULTS
 DISCUSSION
 REFERENCES
 
Brain tumors were induced as described above in Wistar rats using 5 x 105 C6 glioma cells/animal. After tumor formation was detected by MRI, either 4 x 106 {Psi}CRIP-retro-1.3MBPp-luc-transduced cells or the same number of {Psi}CRIP-retro-1.3MBPp-pe-toxin-transduced cells were inoculated at the same stereotactic coordinates used for the initial injection of C6 cells. During each day of the following week, each animal was given 200 µg of T3 (0.4 µg/µl) by i.p. injection.

All rats received tetracycline in drinking water (approximately 75 mg/kg) and dexamethasone (1 mg/500 ml) for 2 days before and after surgery.


   MRI
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Inoculation of Tumor Cells
 Treatment of Induced C6...
 MRI
RESULTS
 DISCUSSION
 REFERENCES
 
The rats were anesthetized with a mixture of ketamine (50 mg/ml), valium (5 mg/ml), and atropine (1 mg/ml) in a 5:4:1 ratio by volume at a dose of 0.3 ml/100 g body weight to obtain the magnetic resonance image. In this study, axial, sagittal, and coronal views of the skull were made with a Cpflex small coil around the animal. Quantitative in vivo measurements included tumor volume and localized T1 and T2 relaxation times. Projection images were obtained from the 4-mm slice acquired with a repetition time (TR) of 420 ms, echo time (TE) of 17 ms, a 256 x 512 matrix, and a 50 x 100 field of view. T1 high-resolution images were obtained after enhancement with gadolinium (2 ml/kg). Tumors appear hyperintense, with distinct tumor margins and only moderate peritumoral edema.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Inoculation of Tumor Cells
 Treatment of Induced C6...
 MRI
 RESULTS
DISCUSSION
 REFERENCES
 
MBPp-driven luc Expression in a Retroviral System.
We have chosen the promoter region of the mouse mbp gene (18) as a possible candidate for glioma-specific transcription initiation. To analyze the tissue specificity conferred by the MBPp, different retroviral plasmids were constructed (retro-1.3MBPp-luc and retro-256MBPp-luc; Fig. 1Citation ). The vectors carried pac as a selection gene and luc as a reporter gene controlled by the 5'-LTR promoter in one case (retro-luc), by the large (1.3-kbp) MBPp in a second one, and by the small 256-bp MBPp in a third type of construct (17) . In the retro-MBPp-luc plasmids, the promoter and gene were placed in opposite sense to retroviral 5'-LTR controlled transcription. C6 cells were transiently transfected with retro-1.3MBPp-luc and retro-256MBPp-luc and assayed for luc activity (Fig. 2ACitation ). As a control, plasmid Y, which does not contain the reporter gene, and plasmid retro-luc, which carries the luc gene under the control of the 5'-LTR promoter, were used. Activity was detected in all samples but the control. The vector retro-1.3MBPp-luc was then transfected to cells with a different tissue origin ({Psi}CRE, rat fibroblast), and after puromycin selection, luc activity was also measured (Fig. 2BCitation ). The large activity estimated shows that in vitro the MBPp does not maintain the cell specificity described in vivo.



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  		Fig. 2. Absence of tissue specificity in T3-dependent activation of the MBPp in vitro. A, luc activity detected in transient transfections of rat glioblastoma C6 cells with the plasmids shown on the abscissa, in the presence of T3. A transfection negative control (Y) and a positive one (retro-luc) are shown. B, luc activity detected in nontransfected (NT) {Psi}CRE cells and in stably transfected mouse fibroblast {Psi} CRE cells in the presence of T3.

 
Regulation of MBPp-driven luc Expression by TRß1/T3.
The role played by T3 in the activation of the MBPp was tested using both transient (Fig. 3ACitation ) and stable transfections (Fig. 3, B and CCitation ). Because the levels of expression of the hormone receptor (TRß1) were unknown, the retroviral LNCXTRß1 or the nonretroviral pRSVTRß1 plasmids containing the TRß1 gene under the control of the CMV or pRSV promoter were cotransfected with retroviral plasmids bearing the MBPp. Regulation of luc expression by the presence of T3 is clearly observed in all cases (Fig. 3ACitation ). No increase in T3 stimulation is observed in cotransfections with the hormone receptor TRß1, indicating the endogenous presence of this molecule in sufficient concentration to maintain the T3-dependent expression. Plasmid retro-1.3MBP-luc shows the greatest stimulation, being slightly better than the smaller (256-bp) MBPp at T3-mediated activation. luc activity was detected even in the absence of T3, probably due to the cell heterogeneity expected in culture cell lawns and transient expression, in which luc-expressing and non-luc-expressing cells are mixed.



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  		Fig. 3. MBPp expression dependent on the presence of T3. A, luc activity detected in transient cotransfections of rat glioblastoma C6 cells in the presence or absence of T3 in the medium. B, luc activity detected in individual clones of {Psi}CRIP cells after stable cotransfection with retro-1.3MBPp-luc and pRSVTRß1 in the presence and absence of T3. C, luc activity detected in individual clones of {Psi}CRIP cells after stable cotransfection with retro-256MBPp-luc and pRSVTRß1 in the presence and absence of T3. The luc activity represented in B and C was the result of single experiment.

 
When packaging cell individual clones were obtained and their luc activities were assayed after growing them for 48 h in the presence or absence of T3, some of them showed a marked repression of the promoter in the absence of T3 (Fig. 3BCitation ). Two clones in particular, clone 7 and especially clone 19, showed a very clear response to T3. Results that were not so excellent but good nonetheless were obtained using the small 256-bp MBPp, indicating that these 256 bp are sufficient to act as a T3 regulatable element (Fig. 3CCitation ).

Effect of T3 on MBPp-regulated Toxin Retrovirus-transduced Cells in Vitro.
Given the tight levels of repression achieved in some clones using the reporter gene, we constructed two new vectors in which the luc gene was replaced by either the pe gene or the ricin gene (Fig. 1CCitation ). Producer cells of retroviruses bearing toxic genes were generated by transduction of the corresponding retroviral plasmid, followed by puromycin selection in the absence of T3. In the presence of T3, it was not possible to obtain a living culture. Antibodies against pe confirmed the expression of the gene product in the presence of T3 and its absence when T3 was removed from the serum (Fig. 4Citation ). The retroviruses produced by these cells were used to infect rat (C6) and human (U-373-MG) glioblastoma cells. After puromycin selection, stable cultures were maintained in the absence of T3. The activation of toxin expression from the MBPp in the presence of T3 was also measured, estimating the death rate of these stable cell lines on the addition of T3-containing serum to the culture medium. A 50% reduction in the incorporation of [3H]thymidine into DNA was observed at 24, 48, 72, and 96 h after T3 treatment in the retro-MBPp-pe-toxin-infected {Psi}CRIP and C6 cells. A 50% reduction was also seen in the human glioblastoma cell line U-373-MG infected with retro-1.3MBPp-ri, and a progressively decreasing reduction of as much as 70% was seen in the retro-MBPp-ri-toxin-infected {Psi}CRIP cells (Table 1)Citation . The results were confirmed with methylene blue staining of live cells. Both toxins are potent inhibitors of protein synthesis and induce apoptosis (27) . When the stable infected {Psi}CRIP-MBPp-pe-toxin and {Psi}CRIP-MBPp-ri-toxin were transferred to T3-containing medium for 24 h and labeled for 4 h with [35S]methionine and [35S]cysteine, a 50% inhibition of protein synthesis was observed (Table 2)Citation . All of the experiments presented here show that the toxins are expressed upon T3 addition to the media in vitro and are able to induce significant cell death in the infected cells.



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  		Fig. 4. Immunofluorescence microscopy of pe. Retro-1.3MBPp-pe-toxin producer {Psi}CRIP cells were cultivated for 48 h in the absence (A) or presence (B) of T3 and labeled with a polyclonal anti-exotoxin A antibody followed by a fluorescein-conjugated antirabbit second antibody. In C and D, retro-1.3 MBPp-pe-toxin-infected C6 cells were cultivated for 48 h in the absence (C) or presence (D) of T3. pe expression was detected as described previously using a Texas red-conjugated antirabbit second antibody.

 

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  		Table 1 Percentage reduction of thymidine incorporation in different stable infected cells when T3 is added to the mediuma

 

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  		Table 2 Percentage of inhibition of protein synthesis induced by T3 in {Psi}CRIP infected cellsa

 
Effect of T3 on MBPp-regulated Toxin Retrovirus-transduced Cells in Vivo.
To evaluate the applicability of the system in vivo, we performed several experiments on SCID mice, normal mice, and Wistar rats.

Neither retro-1.3MBPp-luc- nor retro-1.3MBPp-toxin-infected C6 cells were capable of progressing to form a tumor when injected into the flanks of immunocompetent rats (n = 20). When C6-retro-1.3MBPp-luc cells were injected into the flanks of BALB/c (immunocompetent) mice, a small tumor developed in 3 of 20 inoculations, and no tumors developed in the C6-retro-1.3MBPp-toxin inoculations (Table 3)Citation . Nevertheless, s.c. tumors were easily induced by the injection of infected C6 cells into the lateral flanks of immunodeficient SCID mice. Thus, we injected retro-1.3MBPp-luc-infected C6 cells into the left flank of six mice as a negative control while injecting neoplastic cells infected with either retro-1.3MBPp-pe-toxin (n = 3) or retro-1.3MBPp-ri-toxin (n = 3) into the right side. As shown in Fig. 5Citation and Table 3Citation , all control tumors developed well (Fig. 5Citation , top row), whereas toxin-gene-retrovirus-infected neoplastic cells produced very small tumors or no tumor at all when cells were infected with the pe-retrovirus (as seen in the third pair in Fig. 5ACitation ). Cells infected with the ricin-retrovirus also gave rise to very small tumors (Fig. 5BCitation ; Table 3Citation ), but the effect is not as great as that observed with the pe gene. The difference could be attributed to the presence of the receptor recognition domain in exotoxin A but not in the ricin gene, where only the catalytic polypeptide A has been expressed. The recognition domain is necessary for the interaction and introduction of the toxin into surrounding cells (28) and could account for a bystander effect. In the case of ricin, a higher toxic activity could probably be obtained by coexpression of both the A and B chains.


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  		Table 3 Summary of the in vivo data

 


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  		Fig. 5. s.c. tumors observed in the flanks of SCID mice 10 days after the injection of C6 cells treated in different ways. A, top row, left-side tumors induced after injection of retro-MBPp-luc-infected C6 cells. Bottom row, the corresponding right-side tumors induced by injection of C6 cells infected with the retro-1.3MBPp-pe-toxin vector. B, top row, tumors induced by retro-1.3MBPp-luc-infected C6 cells injected in the left side of the animal; bottom row, corresponding right-side tumors induced by injection of C6 cells infected with the retro-1.3MBPp-ri-toxin vector.

 
In a second set of experiments, retro-1.3MBPp-luc-infected C6 cells were injected into the rat brain and allowed to form a tumor (n = 2). In parallel, other animals (n = 2) were inoculated with retro-1.3MBPp-pe-toxin-infected C6 cells (C6-retro-1.3 MBPp-pe-toxin). The same experiments were performed using the ricin-infected cells. As shown in Fig. 6Citation , retro-1.3MBPp-luc-infected C6 cells produced a large tumor in the control animals (Fig. 6, A and BCitation ), whereas both the retro-1.3MBPp-pe-toxin (Fig. 6, C and DCitation )- and retro-1.3MBPp-ri-toxin (Fig. 6, E and FCitation )-infected C6 cells failed to develop a tumor (Table 3)Citation . This indicates that endogenous T3 activates the MBPp, allowing the expression of the two different toxins that kill the neoplastic cells, preventing tumor formation.



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  		Fig. 6. Gadolinium-enhanced MRI showing different animal performance. Coronal (A) and axial (B) views of a 14-day rat glioblastoma induced by injection of C6 cells infected with retro-1.3MBPp-luc. A large tumor has developed. Coronal (C) and axial (D) views of an animal inoculated with retro-1.3MBPp-pe-toxin-infected C6 cells. No tumor has developed. Coronal (E) and axial (F) views of an animal inoculated with C6 cells infected with retro-1.3MBPp-ri-toxin, which also failed to produce a tumor. Coronal (G) and axial (H) views of a 14-day tumor induced by injection of 5 x 105 C6 cells and coronal (I) and axial (J) views of the same tumor treated with 4 x 106 {Psi}CRIP-retro-1.3MBPp-luc showing no eradication of the growing tumor 1 week later. Coronal (K) and axial (L) views of a tumor induced as described in G and H. M and N, coronal and axial views of the same animal (K and L) 1 week after the injection of 4 x 106 {Psi}CRIP-retro-1.3MBPp-pe-toxin producer cells, showing the total regression of the tumor.

 
In a third set of in vivo experiments that more closely reproduce a possible gene therapy protocol for human patients, brain tumors were induced in Wistar rats with C6 cells (5 x 105 cells) and then treated with 4 x 106 murine {Psi}CRIP-retro-1.3 MBPp-pe-toxin retrovirus-producing cells. In control animals, the induced glioblastomas were treated with the same number of murine {Psi}CRIP-retro-1.3 MBPp-luc cells. The murine retrovirus producer cells had a titer of 106 colony-forming units/ml and were injected at a single location (the same location used for C6 delivery, 4 mm to the right of Bregma and 4 mm in depth from the skull). We used retrovirus-producing cells instead of retrovirus because it is not easy to concentrate the virus into a small volume, and because the half-life of a retrovirus at 37°C is only a few hours (3–6 h). In Fig. 6Citation , a tumor developed in a control animal treated with C6 cells can be seen (Fig. 6, G and HCitation ). The tumor continued to grow after treatment with {Psi}CRIP-retroMBPp-luc cells (Fig. 6, I and JCitation ), and the animal died shortly thereafter. Tumors developed after C6 injection (Fig. 6, K and LCitation ) were nevertheless eradicated after intratumoral injection of 4 x 106 murine {Psi}CRIP-retro-1.3 MBPp-pe-toxin producer cells (Fig. 6, M and NCitation ). In both cases, the endogenous levels of T3 were raised with a daily injection of 200 µg (0.4 µg/µl) of commercial T3. No toxicity was observed in two control animals in which 5 x 106 producer cells ({Psi}CRIP-retro-1.3 MBPp-pe-toxin) were injected into normal brain at the same location used for C6 injections.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Inoculation of Tumor Cells
 Treatment of Induced C6...
 MRI
 RESULTS
 DISCUSSION
REFERENCES
 
Retroviral vectors are widely used to transfer genes of interest into a variety of animal and human cells both in vitro and in vivo (29) . These vectors are able to integrate permanently into the host cell genome (30) . Retroviruses are ideally suited for gene therapy of malignant gliomas developed in the CNS, where the normal cells are already mature and do not divide. Because rapidly growing normal cells are rare in the adult CNS, glioma cells can be specifically transduced with a recombinant retrovirus. The clinical assays performed to date have shown that gene transfer into brain tumors is feasible and is capable of inducing a biologically significant response (31) . However, several limitations to the general approach persist (32) . To address some of these problems, we have studied the use of a regulatable expression system in retrovirus-mediated gene therapy.

Selective transcriptional control sequences provide a tool of significant potential to the gene therapist (10) . We chose a promoter specific for glial tissue, mainly oligodendrocytes and Schwann cells, whose activation and repression are dependent on the presence of T3 and its receptor, TRß1 (33) . Our results, in agreement with those of other authors, show that the tissue specificity is lost in vitro (17, 18, 19) . However, we did observe T3-regulated expression in vitro. As cotransfection experiments have shown, the presence of TRß1 did not affect the expression of the MBPp-regulated reporter gene, indicating that, as other authors have found previously (20) , the endogenous TRß1 levels in some cells are sufficient to maintain a T3-dependent expression from this promoter.

Different experimental transcription systems have shown that activation is often not absolute and that the levels can vary significantly between experimental models or cell lines. These characteristics may be incompatible with the stringent needs of a given therapeutic application (34 , 35) . We have been able to obtain a total repression of a reporter gene after selecting an adequate clone ({Psi}CRIP-retro-1.3MBPp-luc clones 7, 11, 18, and 19). The specificity of cellular promoters inserted in the retroviral genome could be overridden by native viral promoters (36, 37, 38) . We have observed that certain clones lose the T3 control of expression when cultured for extended periods (data not shown), and others do not show regulation at all. There is a small difference in T3 luc control expression between the use of the 1300-bp or the 256-bp MBPps on behalf of the longer one (Fig. 3Citation ), but the 256-bp promoter is sufficient to act as a T3 regulatable element.

When the reporter gene was substituted with a toxic gene, we obtained lawns of transduced packaging producer cells using T3-depleted media. Once the selected puromycin-resistant lawns were transferred to T3-containing media, the maximal cell death registered was 70%, although the surviving cells grew very slowly and in most of cases were unable to produce stable cell lines.

The critical point in the procedure described here is the in vitro culture of retroviral producer cells in which the toxin promoter must be turned off completely, whereas the retroviral promoters should be very active. Cells in which a complete repression of the toxin is not achieved will die in culture. However, long-term culture of the infected cells favors the selection of cells that are unable to express the toxin in a T3-dependent manner. To minimize this problem, cells under puromycin selection were never cultured for longer than 2 months.

Toxins are highly immunogenic molecules (27) , and it is very likely that the system operates indirectly via a bystander effect mediated by an immune reaction against toxin-producing cells. Indeed, our results show that rejection of tumors is more efficient in the brain of immunocompetent rats (Wistar) than in SCID mice flanks, suggesting a putative immunomediated bystander effect. In all of the cases studied, the ricin toxin seemed to be less efficient than Pseudomonas aeruginosa exotoxin A, which was cloned with the eukaryotic receptor recognition domain. Therefore, the success of this gene therapy system in vivo may depend in part on the toxic gene chosen and the orientation of this gene in the retroviral construct.

The gene therapy procedure described in this study can be considered simpler than other killer-suicide systems (1 , 8 , 9) because a single injection of producer cells at the tumor site is sufficient to eliminate the tumor completely, and no side effects have been detected. This system, based on the regulation of toxin gene expression, presents potentially curative applications for the treatment of human brain tumors.


   ACKNOWLEDGMENTS
 
We are indebted to C. O’Kane for kindly providing plasmid pEMBLyex4RA and to I. Pastan for plasmid pVC45F. We also thank Filip Lim for English revision and Marta Vaz for expert technical assistance.


   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 Supported by Comunidad de Madrid Grants 8.1/6/97 and 8.6/21/98, Plan Nacional de Salud Grants 96-37 and PM98-0007, and a grant to the Centro de Biología Molecular from the Fundación Ramón Areces. V. M. was supported by the Ministerio de Educación y Ciencia and the Asociación Española contra el Cáncer. P. d. F. received a grant from the Comunidad Autónoma de Madrid. The work of A. F. was partially supported by an AIRC grant to Alfredo Pontecorvi. Back

2 To whom requests for reprints should be addressed, at Departamento de Biología Molecular, Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Facultad de Ciencias, Cantoblanco 28049 Madrid, Spain. Phone: 00-34-91-3974857; Fax: 00-34-91-3974799; E-mail: mizquierdo{at}cbm.uam.es Back

3 The abbreviations used are: MBP, myelin basic protein; MBPp, MBP promoter; pe, Pseudomonas aeruginosa exotoxin A; ri, chain A of Ricinus communis toxin; T3, thyroid hormone; luc, luciferase; pac, puromycin N-acetyl transferase; TRß1, thyroid receptor ß1; CMV, cytomegalovirus; RSV, Rous sarcoma virus; CNS, central nervous system; SCID, severe combined immunodeficient; LTR, long terminal repeat; MRI, magnetic resonance imaging. Back

Received 11/ 5/99. Accepted 4/19/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Inoculation of Tumor Cells
 Treatment of Induced C6...
 MRI
 RESULTS
 DISCUSSION
 REFERENCES
 

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