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[Cancer Research 61, 6925-6930, September 15, 2001]
© 2001 American Association for Cancer Research

Molecular Biology and Genetics

Self-deleting Suicide Vectors (SDSV)

Selective Killing of p53-deficient Cancer Cells1

Thomas Andreú, Christoph Ebensperger, Eva-Maria Westphal, Thomas Klenner, A. Francis Stewart, Andreas Westhof, Peter Müller, Rainer Knaus and Harald von Melchner2

Laboratory for Molecular Hematology, Department of Hematology, University of Frankfurt Medical School, 60596 Frankfurt [T. A., C. E., E-M. W., H. v. M.]; Department for Cancer Research, Asta-Medica AG, 60314, Frankfurt [T. K., A. W., P. M.]; Gene Expression Program, European Molecular Biology Laboratory, 69117 Heidelberg [A. F. S.]; and MainGen Biotechnologie GmbH, 60314 Frankfurt [R. K.], Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A self-deleting retrovirus vector carrying a herpes simplex virus (HSV)-thymidine kinase suicide gene has been developed to selectively kill cancer cells expressing a dysfunctional p53 tumor suppressor protein. When cells containing functional p53 are infected with the virus, the integrated provirus and the HSV-thymidine kinase gene are deleted from the genome by site-specific recombination (Cre/loxP). In contrast, cells without p53 or cells expressing a DNA-binding mutant of p53 retain the provirus and become susceptible to killing by ganciclovir. This strategy provides a new concept for the selective killing of cancer cells that can be adapted to any other dysfunctional transcription factor expressed by different tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A major goal of cancer research is the development of therapies that can selectively kill tumor cells without adversely affecting normal cells. To this end, several strategies have taken advantage of genetic defects selectively expressed in cancer cells of which the most prominent example is the tumor suppressor gene p53. p53 is mutationally inactivated in >50% of human cancers and is functionally deficient in many other tumors because of mutations in interacting genes (1 , 2) . On the basis of these observations, Onyx-Pharmaceuticals designed a human adenovirus intended to selectively replicate in and kill tumor cells that lack functional p53 while simultaneously remaining nontoxic and replication-defective in cells with an intact p53. This adenovirus, referred to as "Onyx-015," lacks the gene encoding the p53-inactivating E1B55K protein and, therefore, cannot replicate in the presence of normal p53. Yet, replication can occur in the absence of functional p53, resulting in a selective killing of p53-deficient cells (3 , 4) . Onyx-015 has shown activity in preclinical models of cancer and is now undergoing clinical trials in patients with advanced disease (5 , 6) . However, several groups reported recently that replication of Onyx-015 does not correlate with the p53 status of the cells (6, 7, 8, 9, 10, 11) . Moreover, it was concluded by one group that although the virus can replicate in all cells, p53wt3 is required for cytolysis (7) . Whereas the issue remains unresolved, it is likely that other defects upstream or downstream of p53 in the p53 pathway are partly responsible for the conflicting results. Indeed, some Onyx-015-susceptible tumor cells have been shown recently to express a dysfunctional p14ARF-MDM2-p53 pathway resulting in a reduced ability of p53 to activate its target genes (12) .

To develop an alternative strategy that would perhaps be more specific to p53, we constructed a self-deleting retrovirus vector in which deletion from the genome of an infected cell is dependent on transcriptional activation by p53. Self-deleting retrovirus vectors are based on the Cre/loxP site-specific recombination system and contain a Cre-recombinase gene controlled by a virus-independent promoter. In a typical vector a loxP site is inserted in the terminal U3 control region of the retrovirus, which duplicates during replication to form the LTRs. This places the Cre-expressing proviral genome between loxP sites and triggers its excision (13 , 14) .

Because mutations in cancer cells invariably inactivate the ability of p53 to bind DNA, we designed a vector in which Cre expression is dependent on transactivation by p53 so that self-deletion only occurs in cells expressing functional p53. Cells lacking functional p53 would retain the virus and could then be earmarked by a suicide gene.

We show here that SDSVs carrying the HSV-Tk gene and a p53-inducible Cre-recombinase are selectively excised from cells expressing functional p53 (p53wt). Infected cells lacking p53 or expressing a DNA-binding mutant (p53mut) retain the provirus and are killed by the prodrug GCV. Moreover, we demonstrate in an animal model that p53wt protects SDSV-expressing cells from killing by GCV.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids.
The expression plasmid pBSpg13{Delta}MMTVCrepA was obtained by first cloning the Cre coding sequences derived from pU3Cre (15) into the ClaI site of pBluescript KS (Stratagene). The pg13 sequence (16) subcloned into pGL3 (Promega) was recovered as a SacI/EcoRI fragment and cloned into the corresponding sites of pBSCre. The truncated MMTV promoter, {Delta}MMTV, was obtained from p{Delta}MtetO-Luc (17) by PCR using the following MMTV-specific primers: 5-cctatgttcttttggaacttatcc-3 and 5-agggccctgttcgggcgcc-3 and inserted into the HindIII site of pBSCre to obtain pBSpg13{Delta}MMTVCre. Finally, the SV40 polyadenylation sequence from pGL3 was cloned into the SalI site of pBSpg13{Delta}MMTVCre to obtain pBSpg13{Delta}MMTVCrepA.

To obtain the retroviral vector pSDSV{Delta}MMTV, we first inserted HSV-Tk into pBSpg13{Delta}MMTVCrepA. HSV-Tk pg13{Delta}MMTVCre was then cloned into the ClaI site of pBabe (18) and lxSVPuro from pggSVCreU3lxSVpuro (14) into the NheI site of pBabe’s 3'LTR.

The plasmid pSBC2-p53wt was obtained by inserting the coding sequence of p53 into the EcoRI/HindIII sites of the pSBC2 expression vector (19) .

Cells and Viruses.
Except for the U87 cell line, all of the cells were grown DMEM (Life Technologies, Inc.) supplemented with 10% (SAOS-2 and HT-29) or 20% (MCF-7) bovine serum. U87 cells were passaged in MEM (Life Technologies, Inc.) supplemented with 10% FCS (Life Technologies, Inc.). The Cre reporter cell line SAOS-2-CR was obtained by transfecting 10 µg of pSVpaX1 (20) into SAOS-2 cells and by selecting in 0.5 µg/ml puromycin. To assay for p53-inducible Cre expression, 10 µg of pBSpg13{Delta}MMTVCrepA were cotransfected with 10 µg of pSBC-p53wt or 10 µg of empty control vector into SAOS-2CR cells. After 72 h cells were stained with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside and analyzed as described previously (21) .

Helper virus-free recombinant retroviruses were obtained by transient transfection into {Phi}NXampho as described by Yang et al. (22) . Infections were performed by incubating 2 x 105 cells for 16 h with undiluted and filtered viral supernatants in the presence of 8 µg/ml Polybrene (Aldrich). Provirus-containing clones were isolated by selecting for 7 days in medium containing 0.5 µg/ml (SAOS-2) or 2.5 µg/ml (HT-29, MCF-7, U87, and HFBs) puromycin (Sigma Chemical Co.).

p53 induction was performed by exposing SDSV-infected cells to either 1 µM of DXR (Sigma Chemical Co.) for 16 h or to 0.01 µM DXR for 3 h.

VSV-G pseudotyping was performed by cotransfecting Phoenix-gp cells with pSDSV{Delta}MMTV and the VSV-G expression plasmid pHCMV-G, essentially as described previously (23) .

To generate high titers of infectious virus required for the animal experiments, 5 x 107 Phoenix-gp cells were plated onto gelatin-coated 22.5 x 22.5 cm dishes (Corning) and incubated overnight. After adding 25 ml of fresh medium, cells were treated with 5 ml of a calcium-phosphate transfection mix containing 164 µg of pSDSV{Delta}MMTV and 36 µg of pHCMV-G. After incubating overnight and washing once with fresh medium, virus particles were harvested in 25 ml of medium every 12 h for 3 consecutive days. Virus containing supernatants were filtered through a 0.45-µM Millipore filter using a 250-ml Bottle-top-Filter (Millipore). Virus titers were determined on U87 cells by infecting with virus-containing supernatants and selecting in puromycin. SDSV titers varied between 5 x 104 and 1 x 105 particles/ml. Supernatants were concentrated by ultracentrifugation at 26,000 rpm in a Beckman SW-28 rotor for 1.5 h. Virus pellets were resuspended in 2 ml of medium and stored at -80°C until use.

Analysis of Cell Viability.
Cell viability was estimated by using the Cell Proliferation kit (XTT; Roche) according to the manufacturer’s instructions. Briefly, 2000 cells were grown for 5 days in the presence or absence of 25 µM GCV (Syntex), and cell metabolism was measured in a MR5000 (Dynatech) spectrophotometer after incubating with XTT substrate for 2 h.

DNA Isolation and PCR Analysis.
Genomic DNA was isolated by using the QIAamp Tissue kit (Qiagen) and the manufacturer’s instructions. For PCR assays, genomic DNA derived from individual clones was amplified for 33 cycles (94°C for 1 min, 58°C for 1 min, and 72°C for 1 min followed by 72°C for 15 min) using the HSV-Tk-specific primers 5'-atgctggccgcgattcgccgcgttt-3' and 5'-ttatcgataccgtcgactagccttc-3'. For control PCRs the same DNA was amplified for 25 cycles (94°C for 1 min, 60°C for 1 min, and 72°C for 1 min) using the actin-specific primers 5'-gtgacgaggcccagagcaagag-3' and 5'-aggggccggactcatcgtact-3'.

Western Blot Analysis.
Cells were lysed in radioimmunoprecipitation assay buffer supplemented with phenylmethylsulfonyl fluoride (Sigma Chemical Co.), aprotinin (Sigma Chemical Co.), and leupeptin (Sigma Chemical Co.), resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were treated with 1 µg/ml monoclonal anti-p53 antibody (CBL 420; Cymbus Biotechnology) and processed as described in the Research Applications Manual of Santa Cruz Biotechnology.

Animal Experiments.
VSV-G pseudotyped SDSV{Delta}MMTVSDSV retrovirus infections were performed by incubating 1.6 x 105 U87 cells with virus supernatant containing 6.4 x 105 particles (MOI = 4). After incubating overnight, cells were allowed to recover for 2 days in fresh medium. Then the cells were split into equal aliquots. One aliquot was exposed to 0.01 µM DXR for 3 h, after which cells were washed and kept growing in fresh medium for three passages to increase the number of infected cells. This procedure significantly reduced the number of infectious particles required to achieve a high MOI.

Infected U87 cells (5 x 106) were injected s.c. into the flanks of 8-week-old female nude mice (NMRI-nu/nu; Möllegard/Bomholtgaard, Ejby, Denmark). After tumors reached a size of ~0.2 g, groups of eight mice were injected i.p. twice daily with 12.5 mg/kg, 25 mg/kg, or 50 mg/kg GCV (Cymeven; La Roche) for 5 consecutive days, respectively. Control mice received noninfected cells and were similarly treated with 0.9% saline or 50 mg/kg GCV. Tumor size was measured twice/week using standard procedures. All of the animal experiments conformed to German Federal Regulations.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Construction of a Self-deleting Retrovirus Vector Inducible by p53.
To identify a p53-inducible promoter that would tightly control Cre expression, we cloned the -pg13- p53 DNA-binding motif (16 , 24) into Cre expression vectors containing the enhancer-deleted promoters of CMV or MMTV (17 , 25) . p53-inducible Cre expression was tested in SAOS-2-CR human osteosarcoma cells by cotransfecting Cre and p53 expression plasmids. SAOS-2-CR have no endogenous p53 and contain a copy of the Cre reporter plasmid pSVpaX1. pSVpaX1 recombines in the presence of Cre-recombinase, resulting in the expression of ß-galactosidase, which is detectable by histochemical staining (20) . Initial experiments revealed that both minimal promoters stimulated sufficient Cre expression in the absence of p53 to cause pSVpaX1 recombination in >50% of the cells (data not shown).

To minimize this background expression, we made several deletion mutants of both minimal promoters and tested their ability to induce Cre expression with and without p53. A truncated MMTV promoter including the region between nucleotides 1175 and 1417 of the MMTV-LTR (GenBank accession no. J02271) was almost inactive in the absence of p53 but highly inducible by p53 (Fig. 1)Citation .



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  		Fig. 2. Predicted proviral structure and excision mechanism of SDSV. Activation of Cre expression by functional p53 in normal cells excises all proviral sequences flanked by loxP sites from the genome, leaving a single LTR behind. Lack of Cre activation in the absence of functional p53 in cancer cells results in provirus retention and expression of HSV-Tk, thus enabling killing by GCV. lx, loxP; SV, SV40 promoter; Puro, puromycin resistance gene; pg13, p53 binding sequence; {Delta}M, {Delta}MMTV (truncated MMTV promoter); WT, wild type; MUT, DNA-binding mutant.

 
The Cre expression cassette pg13{Delta}MMTVCre was cloned into a Moloney murine leukemia virus vector downstream of an HSV-Tk gene (26) . To enable self-deletion and positive selection of infected cells, an independently expressed puromycin resistance gene flanked by a loxP recombination site was inserted into the U3 region of the 3'LTR (Fig. 2)Citation . As has been shown previously, virus replication and LTR-mediated duplication places the viral genome between two loxP sites and renders it susceptible to excision by Cre recombinase (14) . Thus, we expected that Cre induction by functional p53 would delete the proviral genome, including the genes encoding HSV-Tk and Cre. In contrast, cells without p53 would retain the proviral genome and, by expressing HSV-Tk, would be susceptible to killing by the prodrug GCV (Fig. 2)Citation .

To test this prediction, the final construct, pSDSVTk{Delta}MMTV, was transiently transfected into {Phi}NXampho helper cells (22) to produce infectious virus that was used to infect the human (a) SAOS-2 osteosarcoma cells that lack p53 (p53-/-); (b) HT-29 colon carcinoma cells that express p53mut; and (c) MCF-7 breast carcinoma cells that express normal p53wt.

GCV Kills SDSVTk{Delta}MMTV-expressing Cancer Cells That Lack Functional p53.
Individual SDSVTk{Delta}MMTV-expressing SAOS-2, HT-29, and MCF-7 clones, isolated by selecting in puromycin, were exposed to GCV. Cell viability in each clone was quantified spectrophotometrically using a colorimetric assay (XTT; Ref. 27 ).

As shown in Fig. 3, A and BCitation , cell viability in the presence of GCV was significantly reduced in 15 of 18 SAOS-2 and 20 of 24 HT-29 clones. Whereas variable numbers of surviving cells were still present in individual clones, as one would expect from a brief exposure to GCV (5 days), the increased susceptibility to GCV indicated that a majority (83%) expressed the proviral HSV-Tk gene. To verify its presence in the genome, all clones were subjected to PCR-analysis using HSV-Tk-specific primers. As expected, clones exhibiting reduced viability in GCV produced a specific HSV-Tk amplification product consistent with a provirus being retained by the genome of the cells. In contrast, SAOS-2 and HT-29 clones resistant to GCV lacked the amplification product, indicating that the proviruses had recombined (Fig. 3, A and BCitation Ref. 14 ). Taken together, the results show that the majority of SDSVTk{Delta}MMTV proviruses fail to self-delete in the absence of functional p53. The few proviruses that recombined are most likely the result of low level Cre expression enabled by integrations into transcriptionally active chromosomal regions.



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  		Fig. 3. Selective killing of p53-deficient cells by GCV. XTT survival assay and analysis of recombination in p53-/- SAOS-2 osteosarcoma cells (A), p53mut expressing HT-29 colon carcinoma cells (B), and p53wt expressing MCF-7 breast carcinoma cells (C). Individual clones infected with SDSVTk{Delta}MMTV were isolated by selecting in puromycin and exposed to 25 µM of GCV for 5 days. The percentage of viable cells was determined by comparing the light absorbency measured for each clone in the presence of GCV to that obtained for the same clone in the absence of GCV (= 100%). Note that because of the relatively short exposure to GCV, killing of HSV-Tk-expressing clones was not complete in most cases. Genomic DNA corresponding to 10,000 cells of each clone was subjected to PCR. Amplification reactions were performed using HSV-Tk- (top) or actin- (bottom) specific primers and allowed to proceed for 33 and 25 cycles, respectively. Amplification products were resolved on 1% agarose gels and visualized by staining with ethidium bromide.

 
In contrast to SAOS-2 and HT-29 cells, virtually all of the MCF-7 clones survived in GCV, although viability was not always 100% (Fig. 3C)Citation . Accordingly, provirus-specific amplification products were either missing or significantly reduced, indicating that a majority of cells had lost the provirus (Fig. 3C)Citation .

To estimate the efficiency of provirus deletion, we chose several MCF-7 clones in which proviral recombination was not complete and determined the minimum number of cells still capable of generating a visible provirus-specific amplification product under the PCR conditions used. Similarly treated HT-29 clones served as positive controls. Fig. 4Citation shows that 8–16 times more MCF-7 cells are required for a detectable amplification product than HT-29 cells. Assuming that each HT-29 cell retained the provirus, this suggests that <13% of MCF-7 cells retain the provirus in those clones that showed incomplete recombination.



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  		Fig. 4. Provirus recombination efficiency in MCF-7 clones. Genomic DNA from serial dilutions of MCF-7 clones was subjected to PCR with HSV-Tk-specific primers as described in Fig. 3Citation legend. Similarly treated HT-29 clones were used as nonrecombined, positive controls. Note that under the conditions used, DNA from 156 HT-29 cells sufficed to generate a specific amplification product.

 
Provirus Self-deletion Is Enhanced by p53wt Induction.
Because Cre expression and provirus deletion are most likely dependent on the cellular levels of p53, which is low in most normal cells (2) , we investigated whether provirus deletion from MCF-7 clones could be improved by inducing p53 expression. To this end, cells from several MCF-7 and HT-29 clones were treated for 16 h with 1 µM of DXR, which is a potent p53 inducer (28) . p53 levels and provirus recombination were determined before and after treatment by Western blotting with a panantihuman p53 monoclonal antibody and PCR, respectively. Fig. 5, A and BCitation , shows that p53 levels increase significantly in the presence of DXR in both MCF-7 and HT-29 clones, although only MCF-7 cells underwent increased recombination.



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  		Fig. 5. Stimulation of provirus deletion by DXR. Cells from SDSV expressing MCF-7 (A) and HT-29 (B) clones as well as from pooled HFBs (C) were exposed for 16 h to 1 µM DXR. p53 levels were estimated by Western blotting before and after treatment, using the pan-anti-p53 monoclonal antibody CBL420. Recombination efficiency was estimated by using 10 ng of DNA as template.

 
To test whether p53 induction would also stimulate provirus excision from normal human diploid cells, we infected a mixed population of primary foreskin fibroblasts (HFB) with SDSVTk{Delta}MMTV retrovirus and exposed puromycin-resistant cells to DXR. As shown in Fig. 5CCitation , DXR induces p53 in HFBs by at least 1 order of magnitude, which, as expected, significantly stimulates recombination.

In summary, the results illustrate a dose-response relationship between p53 and provirus deletion and underscore the critical requirement for functional p53. Moreover, the results also show that SDSV deletion can be induced in normal cells that hardly express p53.

Killing of SDSV-infected Tumor Cells by GCV Does Not Require Selection for Provirus Expression.
For most cancers, gene therapy approaches a selection protocol for transduced cells is impractical. To investigate whether SDSVs would also work without selection, several cell lines were infected with VSV-G pseudotyped SDSVTk{Delta}MMTV (23) at high multiplicities of infection (MOI = 4). Subsequent exposure to GCV showed that cells expressing dysfunctional p53 or no p53 are effectively killed even without selection (Table 1)Citation . To investigate whether an extended exposure to GCV would increase the fraction of dying cells, 2000 SDSV-infected SAOS-2 or HT-29 cells were seeded on to 100-mm Petri dishes and incubated for 4 weeks in 25 µM GCV. In each case, <5% of the seeded cells formed visible colonies (100/2000 and 33/2000 for SAOS-2 and HT29, respectively), indicating that the majority of the cells had died.


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  		Table 1 Summary of results obtained with SDSVs and nonselected cell pools

 
In contrast, cells expressing p53wt were mostly GCV-resistant except for normal human fibroblasts (Table 1)Citation . Of the latter, only 50% survived in GCV, presumably because of borderline expression of p53 (Fig. 5C)Citation . However, pretreatment with DXR nearly doubled the number of surviving cells, suggesting that almost complete SDSV deletion can be induced in cells with minimal expression of p53 (Table 1)Citation .

In Vivo Suicide of SDSV-infected Tumor Cells Is Dependent on the Levels of p53wt.
To investigate whether an SDSV approach would be suitable in vivo, we first examined the ability of the cell lines to form tumors in immunodeficient (nude) mice. Because none of the different cell types were comparable in terms of growth kinetics or resistance to GCV in vivo, we focused on one cell line that expresses p53wt, i.e., U87.

To test whether p53 levels correlate with tumor formation in GCV-treated mice, U87 cells were infected with VSV-G pseudotyped SDSVTk{Delta}MMTV retrovirus at a high MOI (MOI = 4) and transplanted s.c. into the flanks of nude mice. Before transplantation, aliquots were treated for 3 h with 0.01 µM of DXR to induce p53. This regimen caused significant p53 induction and was well tolerated by the cells (data not shown). In this context, it is of interest that a single application of a nontoxic dose of DXR (1.75 mg/kg) in vivo resulted in a significant (up to 5-fold) and sustained (up to 17 days) elevation of p53 levels in mouse liver cells (data not shown). Thus, DXR appears to be an effective p53 inducer in vitro and in vivo.

To accurately compare tumor formation, 5 x 106 DXR-pretreated or untreated cells were transplanted such that each mouse carried a p53-induced xenograft on the left side and a p53-uninduced graft on the right side. After tumors developed to a size of ~0.2 g, groups of mice received twice daily 12.5–50 mg/kg body weight GCV for 5 consecutive days. Control mice with uninfected xenografts received either saline or 50 mg/kg GCV. Whereas DXR had no effect on tumors grown from uninfected cells (Fig. 6, A and B)Citation , it significantly stimulated growth of tumors derived from the infected cells. Fig. 6 (C–F)Citation shows that in groups receiving GCV, DXR-pretreated xenografts (left flank) formed tumors that were 3–5 times larger than tumors developing from the untreated xenografts (right flank), strongly suggesting that proliferation is dependent on the levels of p53.



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  		Fig. 6. Tumor formation by SDSV-infected U87 cells in mice treated with GCV. Groups of six to eight mice were injected s.c. with 5 x 106 SDSVTk{Delta}MMTV-infected U87 cells, which were either pretreated (left flank) or not treated (right flank) with DXR. DXR treatment was applied for 3 h at a concentration of 0.01 µM. A and B, control mice transplanted with noninfected U87 cells and treated with 0.9% saline (A) or 50 mg/kg GCV (B). C–E, mice transplanted with SDSV-infected U87 cells with i.p. injections of 12.5 mg/kg (C), 25 mg/kg (D), and 50 mg/kg (E) GCV (Cymeven), respectively. All injections were applied twice daily for 5 consecutive days. F, examples of asymmetric tumor growth. Featured animals are identified by numbers. Colors correspond to individual mice. The significance of differences between left and right tumor sizes on day 42 was estimated by the two-tailed t test. Calculated Ps were as follows: groups 1 and 2, not significant; group 3, P = 0.01; group 4, P < 0.0001; group 5, P = 0.005.

 
Taken together, the results provide an in vivo proof for the SDSV concept and suggest that selectivity achieved by Cre/loxP recombination could be useful in the development of alternative strategies for the gene therapy of cancer.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have used the Cre/loxP site-specific recombination system to design a retrovirus vector that discriminates between cells expressing functional or dysfunctional p53. Transduction of a p53 controlled Cre recombinase in conjunction with a constitutively expressed HSV-Tk suicide gene into several types of human cancer cells resulted in a p53-dependent excision of the transduced genes. Cells lacking functional p53 failed to eliminate the suicide gene and were killed selectively by GCV. Moreover, selective resistance to GCV could be significantly improved in vitro and in vivo by inducing p53. Finally, the in vivo studies clearly demonstrated a link between p53 levels and GCV killing of SDSV infected cells.

By exploiting the ability of p53 to bind DNA and act as a transcription factor, the self-deleting suicide vectors could provide an alternative to existing concepts of cancer treatment. Alone or in combination with p53 inducing drugs, the vectors are likely to enable selective killing of cancer cells while having little or no toxic effects on normal cells. Because toxicity is dependent on the levels of p53 in normal cells, which are generally low (Fig. 5)Citation , it is important to emphasize that Cre is an extremely effective recombinase that does not require high level induction to cause recombination (21) . This may explain why a few proviruses recombined in the absence of p53, most likely as an occasional consequence of Cre induction from provirus integrations into transcriptionally active genomic regions. However, this should not constitute a major problem, because both previous studies and our own results indicate that in most solid tumors HSV-Tk-phosphorylated GCV can kill adjacent cells by simply spreading through gap junctions (29) .

Whereas Cre/loxP strategies in combination with tissue-specific promoters have been used previously for the selective killing of cancer cells, all of them require at least two separate viruses. This reduces selectivity, since not all cells will receive a rightly balanced number of both viruses (30, 31, 32) .

Several features of the self-deleting suicide vectors are particularly attractive. First, they are not restricted to p53 mutations and can be adapted to a variety of other transcription factors that are dysfunctional in cancer cells (33) . Second, suicide genes can be easily replaced by genes encoding toxins or proapoptotic proteins of mammalian, bacterial, viral or synthetic origins (34, 35, 36, 37) . Alternatively, genes encoding powerful antigens may be used to induce a cytotoxic immune response against the infected cancer cells. Such a response may also increase the presentation of tumor specific antigens, which would expand the immunological target to noninfected cancer cells (38 , 39) . Third, the self-deleting suicide vectors could be made replication competent, in which case infection would be productive in the tumors but self-limited in normal cells. Finally, the strategy described can be adapted to any other vector system. Particularly useful would be vectors that replicate episomally and, thus, avoid potentially hazardous side-effects of retrovirus integrations such as the activation of cellular oncogenes.

In conclusion, the present paper described a new concept for the gene therapy of cancer, which is selective for transformed cells and flexible enough to be developed for the treatment of a wide range of human cancers.



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  		Fig. 1. Activation of Cre expression by p53. The p53 binding motif pg13 was cloned into the expression vector pBSCre containing the coding sequences for Cre recombinase and a SV40 polyadenylation sequence. A truncated MMTV promoter containing only 242 nucleotides of the LTR was inserted downstream of pg13. The plasmid (10 µg) pBS{Delta}MMTVCrepA was cotransfected with 10 µg of pSBC2-p53wt or 10 µg of empty control vector into SAOS-2CR cells. After 72 h, Cre-mediated recombination of the reporter plasmid pSVpaX1 was analyzed by 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining. In most experiments, the frequency of LacZ positive cells was >30% in cultures cotransfected with pSBC2-p53wt. It was <2% in cultures cotransfected with the empty control vector.

 

   ACKNOWLEDGMENTS
 
We thank Anke Voigt for outstanding technical assistance during the animal experiments.


   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 work was supported by grants from the Bundesministerium für Bildung und Forschung (BMBF; to H. v. M.). Back

2 To whom requests for reprints should addressed, at Laboratory for Molecular Hematology, University of Frankfurt Medical School, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. Phone: 49-69-63016696; Fax: 49-69-63016390; Back

3 The abbreviations used are: p53wt, wild-type p53; LTR, long terminal repeat; SDSV, self-deleting suicide vector; GCV, ganciclovir; p53mut, mutant p53; MMTV, mouse mammary tumor virus; Tk, thymidine kinase; MOI, multiplicity of infection; DXR, doxorubicin; VSV-G, vesicular stomatitis virus glycoprotein; HFB, human foreskin fibroblast; HSV, herpes simplex virus; Cre, Cre-recombinase; loxP, Cre-recombinase recognition sequence; VSV, vesicular stomatitis virus; CMV, cytomegalovirus. Back

Received 4/ 2/01. Accepted 7/17/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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V. Dixit and R. L. Juliano
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