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Experimental Therapeutics |
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.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSInoculation of Tumor CellsTreatment of Induced C6...MRIRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSInoculation of Tumor CellsTreatment of Induced C6...MRIRESULTSDISCUSSIONREFERENCES |
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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
(
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.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSInoculation of Tumor CellsTreatment of Induced C6...MRIRESULTSDISCUSSIONREFERENCES |
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CRE cells and amphotropic
retroviral packaging 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. 1
) 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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CRE cells and subsequent
harvesting of the supernatant carrying the viral particles to infect
the amphotropic packaging cell line CRIP, according to a method
described previously (26)
. The supernatants of the
transduced 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.
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Inoculation of Tumor Cells |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSInoculation of Tumor CellsTreatment of Induced C6...MRIRESULTSDISCUSSIONREFERENCES |
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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 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.
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Treatment of Induced C6 Wistar Rat Brain Tumors with
CRIP-retro-1.3MBPp-pe-toxin Producer Cells
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSInoculation of Tumor CellsTreatment of Induced C6...MRIRESULTSDISCUSSIONREFERENCES |
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CRIP-retro-1.3MBPp-luc-transduced
cells or the same number of
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.
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MRI |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSInoculation of Tumor CellsTreatment of Induced C6...MRIRESULTSDISCUSSIONREFERENCES |
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSInoculation of Tumor CellsTreatment of Induced C6...MRIRESULTSDISCUSSIONREFERENCES |
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). 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. 2A
). 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 (CRE, rat fibroblast), and after puromycin
selection, luc activity was also measured (Fig. 2B
). The
large activity estimated shows that in vitro the MBPp does
not maintain the cell specificity described in vivo.
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) and stable transfections (Fig. 3, B and C
). 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. 3A
). 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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). 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. 3C
).
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. 1C
). 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. 4
). 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 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 CRIP cells (Table 1)
. 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
CRIP-MBPp-pe-toxin and 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)
. 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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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)
. 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. 5
and Table 3
, all control tumors developed well (Fig. 5
, 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. 5A
). Cells infected with the ricin-retrovirus also gave rise
to very small tumors (Fig. 5B
; Table 3
), 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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, retro-1.3MBPp-luc-infected C6 cells produced a large tumor
in the control animals (Fig. 6, A and B
), whereas
both the retro-1.3MBPp-pe-toxin (Fig. 6, C and D
)- and retro-1.3MBPp-ri-toxin (Fig. 6, E and F
)-infected C6 cells failed to develop a
tumor (Table 3)
. 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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CRIP-retro-1.3
MBPp-pe-toxin retrovirus-producing cells. In control
animals, the induced glioblastomas were treated with the same number of
murine 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. 6
, a tumor developed in a
control animal treated with C6 cells can be seen (Fig. 6, G and H
). The tumor continued to grow after treatment with
CRIP-retroMBPp-luc cells (Fig. 6, I and J
), and the animal died shortly thereafter. Tumors developed
after C6 injection (Fig. 6, K and L
) were
nevertheless eradicated after intratumoral injection of 4
x 106 murine CRIP-retro-1.3
MBPp-pe-toxin producer cells (Fig. 6, M and N
). 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 (CRIP-retro-1.3
MBPp-pe-toxin) were injected into normal brain at the same
location used for C6 injections. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSInoculation of Tumor CellsTreatment of Induced C6...MRIRESULTSDISCUSSIONREFERENCES |
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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
(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. 3
), 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. 
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
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.
Received 11/ 5/99. Accepted 4/19/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSInoculation of Tumor CellsTreatment of Induced C6...MRIRESULTSDISCUSSIONREFERENCES |
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1 and ß 1. J. Biol. Chem., 273: 24239-24248, 1998.This article has been cited by other articles:
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