Genetic polymorphism in Methylenetetrahydrofolate Reductase chloride transport protein 6 (MTHFR CLCN6) gene is associated with keratinocyte skin cancer in a cohort of renal transplant recipients.

Background: Renal transplant recipients (RTRs) are at increased risk of keratinocyte cancer (KC), especially cutaneous squamous cell carcinoma (cSCC). Previous studies identified a genetic variant of the Methylenetetrahydrofolate Reductase (MTHFR) gene, C677T, which conferred a risk for diagnosis of cSCC in Irish RTRs. Objective: We sought to find further genetic variation in MTHFR and overlap genes that may be associated with a diagnosis of KC in RTRs. Methods: Genotyping of a combined RTR population (n = 821) from two centres, Ireland (n = 546) and the USA (n = 275), was performed. This included 290 RTRs with KC and 444 without. Eleven single nucleotide polymorphisms (SNPs) in the MTHFR gene and seven in the overlap gene MTHFR Chloride transport protein 6 (CLCN6) were evaluated and association explored by time to event analysis (from transplant to first KC) using Cox proportional hazards model. Results: Polymorphism at MTHFR CLCN6 (rs9651118) was significantly associated with KC in RTRs (HR 1.50, 95% CI 1.17-1.91, p < 0.00061) and cSCC (HR 1.63, 95% CI 1.14-2.34, p = 0.007). A separate SNP, MTHFR C677T, was also significantly associated with KC in the Irish population (HR 1.31, 95% CI 1.05-1.63, p = 0.016), but not American RTRs. Conclusions: We report the association of a SNP in the MTHFR overlap gene, CLCN6 and KC in a combined RTR population. While the exact function of CLCN6 is not known, it is proposed to be involved in folate availability. Future applications could include incorporation in a polygenic risk score for KC in RTRs to help identify those at increased risk beyond traditional risk factor assessment.

α-PD-1 therapy elevates Treg/Th balance and increases tumor cell pSmad3 that are both targeted by α-TGFß antibody to promote durable rejection and immunity in squamous cell carcinomas.

BACKGROUND: Checkpoint blockade immunotherapy has improved metastatic cancer patient survival, but response rates remain low. There is an unmet need to identify mechanisms and tools to circumvent resistance. In human patients, responses to checkpoint blockade therapy correlate with tumor mutation load, and intrinsic resistance associates with pre-treatment signatures of epithelial mesenchymal transition (EMT), immunosuppression, macrophage chemotaxis and TGFß signaling. METHODS: To facilitate studies on mechanisms of squamous cell carcinoma (SCC) evasion of checkpoint blockade immunotherapy, we sought to develop a novel panel of murine syngeneic SCC lines reflecting the heterogeneity of human cancer and its responses to immunotherapy. We characterized six Kras-driven cutaneous SCC lines with a range of mutation loads. Following implantation into syngeneic FVB mice, we examined multiple tumor responses to α-PD-1, α-TGFß or combinatorial therapy, including tumor growth rate and regression, tumor immune cell composition, acquired tumor immunity, and the role of cytotoxic T cells and Tregs in immunotherapy responses. RESULTS: We show that α-PD-1 therapy is ineffective in establishing complete regression (CR) of tumors in all six SCC lines, but causes partial tumor growth inhibition of two lines with the highest mutations loads, CCK168 and CCK169. α-TGFß monotherapy results in 20% CR and 10% CR of established CCK168 and CCK169 tumors respectively, together with acquisition of long-term anti-tumor immunity. α-PD-1 synergizes with α-TGFß, increasing CR rates to 60% (CCK168) and 20% (CCK169). α-PD-1 therapy enhances CD4 + Treg/CD4 + Th ratios and increases tumor cell pSmad3 expression in CCK168 SCCs, whereas α-TGFß antibody administration attenuates these effects. We show that α-TGFß acts in part through suppressing immunosuppressive Tregs induced by α-PD-1, that limit the anti-tumor activity of α-PD-1 monotherapy. Additionally, in vitro and in vivo, α-TGFß acts directly on the tumor cell to attenuate EMT, to activate a program of gene expression that stimulates immuno-surveillance, including up regulation of genes encoding the tumor cell antigen presentation machinery. CONCLUSIONS: We show that α-PD-1 not only initiates a tumor rejection program, but can induce a competing TGFß-driven immuno-suppressive program. We identify new opportunities for α-PD-1/α-TGFß combinatorial treatment of SCCs especially those with a high mutation load, high CD4+ T cell content and pSmad3 signaling. Our data form the basis for clinical trial of α-TGFß/α-PD-1 combination therapy (NCT02947165).

Interactions between wild-type and mutant Ras genes in lung and skin carcinogenesis.

Ras oncogenes (Hras, Kras and Nras) are important drivers of carcinogenesis. However, tumors with Ras mutations often show loss of the corresponding wild-type (WT) allele, suggesting that proto-oncogenic forms of Ras can function as a suppressor of carcinogenesis. In vitro studies also suggest that WT Ras proteins can suppress the tumorigenic properties of alternate mutant Ras family members, but in vivo evidence for these heterologous interactions is lacking. We have investigated the genetic interactions between different combinations of mutant and WT Ras alleles in vivo using carcinogen-induced lung and skin carcinogenesis in mice with targeted deletion of different Ras family members. The major suppressor effect of WT Kras is observed only in mutant Kras-driven lung carcinogenesis, where loss of one Kras allele led to increased tumor number and size. Deletion of one Hras allele dramatically reduced the number of skin papillomas with Hras mutations, consistent with Hras as the major target of mutation in these tumors. However, skin carcinoma numbers were very similar, suggesting that WT Hras functions as a suppressor of progression from papillomas to invasive squamous carcinomas. In the skin, the Kras proto-oncogene functions cooperatively with mutant Hras to promote papilloma development, although the effect is relatively small. In contrast, the Hras proto-oncogene attenuated the activity of mutant Kras in lung carcinogenesis. Interestingly, loss of Nras increased the number of mutant Kras-induced lung tumors, but decreased the number of mutant Hras-induced skin papillomas. These results show that the strongest suppressor effects of WT Ras are only seen in the context of mutation of the cognate Ras protein, and only relatively weak effects are detected on tumor development induced by mutations in alternative family members. The data also underscore the complex and context-dependent nature of interactions between proto-oncogenic and oncogenic forms of different Ras family members during tumor development.

Hipk2 cooperates with p53 to suppress γ-ray radiation-induced mouse thymic lymphoma.

A genome-wide screen for genetic alterations in radiation-induced thymic lymphomas generated from p53+/- and p53-/- mice showed frequent loss of heterozygosity (LOH) on chromosome 6. Fine mapping of these LOH regions revealed three non-overlapping regions, one of which was refined to a 0.2 Mb interval that contained only the gene encoding homeobox-interacting protein kinase 2 (Hipk2). More than 30% of radiation-induced tumors from both p53+/- and p53-/- mice showed heterozygous loss of one Hipk2 allele. Mice carrying a single inactive allele of Hipk2 in the germline were susceptible to induction of tumors by γ-radiation, but most tumors retained and expressed the wild-type allele, suggesting that Hipk2 is a haploinsufficient tumor suppressor gene for mouse lymphoma development. Heterozygous loss of both Hipk2 and p53 confers strong sensitization to radiation-induced lymphoma. We conclude that Hipk2 is a haploinsufficient lymphoma suppressor gene.

Guidelines for the welfare and use of animals in cancer research.

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

Presenilin modulates EGFR signaling and cell transformation by regulating the ubiquitin ligase Fbw7.

The epidermal growth factor receptor (EGFR) and Notch signaling pathways have antagonistic roles during epidermal differentiation and carcinogenesis. The molecular mechanisms regulating the crosstalk between EGFR and Notch during epidermal transformation are largely unknown. We found enhanced EGFR-dependent signaling, proliferation and oncogenic transformation caused by loss of presenilins (PS), the catalytic components of gamma-secretase that generates the Notch1 intracellular domain (NICD). The underlying mechanism for abnormal EGFR signaling in PS-deficient cells involves gamma-secretase-independent transcriptional upregulation of the E3 ubiquitin ligase Fbw7. Fbw7alpha, which targets NICD for degradation, regulates positively EGFR by affecting a proteasome-dependent ubiquitination step essential for constitutive degradation and stability of EGFR. To investigate the pathological relevance of this findings in vivo, we generated a novel epidermal conditional PS-deficient (ePS cDKO) mouse by deleting both PS in keratinocytes of the basal layer of the epidermis. The ePS cDKO mice develop epidermal hyperplasia associated with enhanced expression of both EGFR and Fbw7 and reduced NICD levels in keratinocytes. These findings establish a novel role for PS on epidermal growth and transformation by reciprocally regulating the EGFR and Notch signaling pathways through Fbw7.

An HDAC1-binding domain within FATS bridges p21 turnover to radiation-induced tumorigenesis.

There is a gap between the initial formation of cells carrying radiation-induced genetic damage and their contribution to cancer development. Herein, we reveal a previously uncharacterized gene FATS through a genome-wide approach and demonstrate its essential role in regulating the abundance of p21 in surveillance of genome integrity. A large exon coding the NH2-terminal domain of FATS, deleted in spontaneous mouse lymphomas, is much more frequently deleted in radiation-induced mouse lymphomas. Its human counterpart is a fragile site gene at a previously identified loss of heterozygosity site. FATS is essential for maintaining steady-state level of p21 protein and sustaining DNA damage checkpoint. Furthermore, the NH2-terminal FATS physically interacts with histone deacetylase 1 (HDAC1) to enhance the acetylation of endogenous p21, leading to the stabilization of p21. Our results reveal a molecular linkage between p21 abundance and radiation-induced carcinogenesis.

Atm heterozygosity does not increase tumor susceptibility to ionizing radiation alone or in a p53 heterozygous background.

Ataxia-Telangiectasia (A-T) is an autosomal recessive human disease characterized by genetic instability, radiosensitivity, immunodeficiency and cancer predisposition, because of mutation in both alleles of the ATM (ataxia-telangiectasia mutated) gene. The role of Atm heterozygosity in cancer susceptibility is controversial, in both human and mouse. Earlier studies identified deletions near the Atm gene on mouse chromosome 9 in radiation-induced lymphomas from p53 heterozygous mice. To determine whether Atm was the target of these deletions, Atm heterozygous as well as Atm/P53 double heterozygous mice were treated with ionizing radiation. There were no significant differences in tumor latency, progression and lifespan after gamma-radiation in Atm heterozygous mice compared with their wild-type control counterparts. Deletions were found on chromosome 9 near the Atm locus in radiation-induced tumors, but in 50% of the cases the deletion included the knockout allele, and the expression of Atm was maintained in the tumors indicating that loss of heterozygosity on chromosome 9 is not driven by Atm, but by an alternative tumor suppressor gene located near Atm on this chromosome. We conclude that Atm heterozygosity does not confer an increase in tumor susceptibility in this context.

Convergence of congenic mapping and allele-specific alterations in tumors for the resolution of the Skts1 skin tumor susceptibility locus.

Although several familial cancer genes with high-penetrance mutations have been identified, the major genetic component of susceptibility to sporadic cancers is attributable to low-penetrance alleles. These 'weak' tumor susceptibility genes do not segregate as single Mendelian traits and are therefore difficult to find in studies of human populations. Previously, we have proposed that a combination of germline mapping and analysis of allele-specific imbalance in tumors may be used to refine the locations of susceptibility genes using mouse models of cancer. Here, we have used linkage analysis and congenic mouse strains to map the major skin tumor susceptibility locus Skts1 within a genetic interval of 0.9 cM on proximal chromosome 7. This interval lies in an apparent recombination cold spot, and corresponds to a physical distance of about 15 Mb. We therefore, used patterns of allele-specific imbalances in tumors from backcross and congenic mice to refine the location of Skts1. We demonstrate that this single tumor modifier locus has a dramatic effect on the allelic preference for imbalance on chromosome 7, with at least 90% of tumors from the congenics showing preferential gain of markers on the chromosome carrying the susceptibility variant. Importantly, these alterations enabled us to refine the location of Skts1 at higher resolution than that attained using the congenic mice. We conclude that low-penetrance susceptibility genes can have strong effects on patterns of allele-specific somatic genetic changes in tumors, and that analysis of the directionality of these somatic events provides an important and rapid route to identification of germline genetic variants that confer increased cancer risk.

Forty years of cancer modelling in the mouse.

Mouse models of human cancer have played an important role in formulating modern concepts of multistage carcinogenesis, and are providing us with a new armoury of tools for the testing of novel therapeutic approaches to cancer treatment. The development of inducible and conditional technologies provide us with greater opportunity to generate mouse models which faithfully recapitulate human tumorigenesis, in terms of both the biology and the genetics of this disease. It is now feasible to control, in time and space, the development of tumours in almost any mouse tissue, such that we now have available mouse models of all major human cancers. Moreover, novel non-invasive approaches to tumour imaging will enable us to follow tumour development and metastasis in vivo, as well as the effects of candidate therapeutic drugs. Such new generation tumour models, which accurately emulate the disease state in situ, should provide a useful platform with which to experimentally test drugs targeted to specific gene products, or combinations of genes that control rate-limiting steps of tumour development.

Building 'validated' mouse models of human cancer.

As a model system for the understanding of human cancer, the mouse has proved immensely valuable. Indeed, studies of mouse models have helped to define the nature of cancer as a genetic disease and demonstrated the causal role of genetic events found in tumors. As the scientific and medical community's understanding of human cancer becomes more sophisticated, however, limitations and potential weaknesses of existing models are revealed. How valid are these murine models for the understanding and treatment of human cancer? The answer, it appears, depends on the nature of the research requirement. Certain models are better suited for particular applications. Using novel molecular tools and genetic strategies, improved models have recently been described that accurately mimic many aspects of human cancer.

TGF-beta signaling in tumor suppression and cancer progression.

Epithelial and hematopoietic cells have a high turnover and their progenitor cells divide continuously, making them prime targets for genetic and epigenetic changes that lead to cell transformation and tumorigenesis. The consequent changes in cell behavior and responsiveness result not only from genetic alterations such as activation of oncogenes or inactivation of tumor suppressor genes, but also from altered production of, or responsiveness to, stimulatory or inhibitory growth and differentiation factors. Among these, transforming growth factor beta (TGF-beta) and its signaling effectors act as key determinants of carcinoma cell behavior. The autocrine and paracrine effects of TGF-beta on tumor cells and the tumor micro-environment exert both positive and negative influences on cancer development. Accordingly, the TGF-beta signaling pathway has been considered as both a tumor suppressor pathway and a promoter of tumor progression and invasion. Here we evaluate the role of TGF-beta in tumor development and attempt to reconcile the positive and negative effects of TGF-beta in carcinogenesis.

Role of matrix metalloproteinase-9 in progression of mouse skin carcinogenesis.

Invasion of malignant tumor cells is required for the formation of metastatic colonies. Uncontrolled expression of matrix metalloproteinase (MMP)-2 and MMP-9 is a critical part of the invasive potential of tumor cells and is affected by the balance between the enzymes and the inhibitors secreted by the cell. Here we analyzed the expression and activity of the two gelatinases (MMP-2 and MMP-9) as well as the expression levels of the tissue inhibitor of metalloproteinase (TIMP2)-, in different stages of carcinogenesis using mouse skin cell lines derived from tumors induced by chemical mutagens. Our results suggested that the expression of MMP-9 was implicated in the progression to spindle cell carcinomas in mouse keratinocytes. MMP-2 levels remained steady in all cell lines, whereas levels of TIMP-2 were increased in normal and spindle cells. The AP-1 DNA binding and transcriptional activity on the MMP-9 promoter were increased in the malignant cell lines, indicating the requirement of this binding site for its activation. The results of this study clearly suggested the important role of MMP-9, but not of MMP-2, in the metastatic properties of mouse keratinocytes.

Replication and cytolysis of an E1B-attenuated adenovirus in drug-resistant ovarian tumour cells is associated with reduced apoptosis.

Therapeutic approaches which are effective in tumour cells resistant to conventional chemotherapy would be of value. An E1B 55 kDa-deleted adenovirus (ONYX-015) induces lysis in cells with mutant p53, although the specificity of these observations for different cell types is unclear. We have used a matched set of drug-resistant human ovarian tumour cell lines to examine the potential of ONYX-015 for preferential replication and lysis of drug-resistant ovarian tumour cells with documented alterations in p53 function. Marked preferential replication of ONYX-015 is observed after infection of mutant p53 transfectant and cisplatin-resistant derivatives, compared to the wild-type p53 expressing parental A2780 line. Infection causes increased cytopathic effects in vitro and inhibition of tumour growth in vivo of the drug-resistant derivatives, but not the parental line. In apparent contrast, increased apoptosis and reduced clonogenic survival is induced by ONYX-015 infection of the chemosensitive parental cell line. ONYX-015 induces increased pro-apoptotic BAX and reduced anti-apoptotic BCLX(L) in parental cells, but not in the resistant derivative A2780/cp70. We propose that induction of apoptosis is one factor which prevents ONYX-015 spread and cytolysis after infection of chemosensitive cells, while it is the failure to engage apoptosis in drug-resistant cells that allows preferential viral replication, spread and cytolysis.

Epistatic interactions between skin tumor modifier loci in interspecific (spretus/musculus) backcross mice.

The development of cancer is influenced both by exposure to environmental carcinogens and by the host genetic background. Epistatic interactions between genes are important in determining phenotype in plant and animal systems and are likely to be major contributors to cancer susceptibility in humans. Several tumor modifier loci have been identified from studies of mouse models of human cancer, and genetic interactions between modifier loci have been detected by genome scanning using recombinant congenic strains of mice (R. Fijneman et al., Nat. Genet., 14: 465-467, 1996; T. van Wezel et al., Nat. Genet., 14: 468-470, 1996; W. N. Frankel et al., Nat. Genet., 14, 371-373, 1996). We demonstrate here that strong genetic interactions between skin tumor modifier loci can be detected by hierarchical whole genome scanning of a complete interspecific backcross [outbred Mus spretus X Mus musculus (NIH/Ola)]. A locus on chromosome 7 (Skts1) showed a highly significant interaction with Skts5 on chromosome 12 (P < 10(-16)), whereas additional significant interactions were detected between loci on chromosomes 4 and 5, and 16 and 15. Some of these quantitative trait loci and their interactions, in particular the Skts1-Skts5 interaction, were confirmed in two completely independent backcrosses using inbred spretus strains (SEG/Pas and SPRET/Ei) and NIH/Ola. These results, therefore, illustrate the general use of interspecific crosses between Mus musculus and Mus spretus for the detection of strong genetic interactions between tumor modifier genes.

Cancer genetics: from Boveri and Mendel to microarrays.

The human genome has now been sequenced, a century after the re-discovery of Mendel's Laws, and the publication of Theodor Boveri's chromosomal theory of heredity. Tracing the historical landmarks of cancer genetics from these early days to the present time not only gives us an appreciation of how far we have come, but also emphasizes the challenges that we face if we are to unravel the genetic basis of hereditary and sporadic cancers in the next century.

TGF-beta inhibits p70 S6 kinase via protein phosphatase 2A to induce G(1) arrest.

On TGF-beta binding, the TGF-beta receptor directly phosphorylates and activates the transcription factors Smad2/3, leading to G(1) arrest. Here, we present evidence for a second, parallel, TGF-beta-dependent pathway for cell cycle arrest, achieved via inhibition of p70(s6k). TGF-beta induces association of its receptor with protein phosphatase-2A (PP2A)-Balpha. Concomitantly, three PP2A-subunits, Balpha, Abeta, and Calpha, associate with p70(s6k), leading to its dephosphorylation and inactivation. Although either pathway is sufficient to induce G(1) arrest, abrogation of both, the inhibition of p70(s6k), and transcription through Smad proteins is required for release of epithelial cells from TGF-beta-induced G(1) arrest. TGF-beta thereby modulates the translational and posttranscriptional control of cell cycle progression.

Allele-specific loss or imbalance of chromosomes 9, 15, and 16 in B-cell tumors from interspecific F1 hybrid mice carrying Emu-c-myc or N-myc transgenes.

Mice carrying an immunoglobulin enhancer (Emu-) linked c- or N-myc transgene develop fatal monoclonal or oligoclonal pre-B or B-cell lymphomas. This indicates that, beside the Emu-activated myc gene, additional genetic changes are required for tumor development. To trace these additional changes, we carried out a genome-wide search for loss of heterozygosity (LOH) and allelic imbalance (AI). This was done at 53 microsatellite markers in a panel of 34 lymphomas and four plasmacytomas from c- or N-myc transgene carrying (BALB/c x Mus spretus)F1 hybrids. An additional 43 lymphomas and three plasmacytomas from non-transgenic F1 mice were also investigated. Losses of one or more spretus-derived chromosome 9 markers were detected in 19 of 23 (83%) of the lymphomas, but in none of the four plasmacytomas that developed in N-myc F1 mice. No LOH-9 was found in any of the 11 lymphomas from Emu-c-myc F1 mice and only in 1 of 46 (2%) tumors derived from non-transgenic (BALB/c x spretus)F1 hybrid controls. These results suggest that a gene on spretus chromosome 9 confers resistance to the development of N-myc but not c-myc-induced lymphomas. AI of chromosome 15 markers (AI-15) was detected in 57 of 77 (74%) lymphomas and in 5 of 7 (72%) plasmacytomas, independently of the transgenic status and the mode of induction. All of the lymphomas and plasmacytomas with AI-15 revealed a relative gain of the spretus-derived D15Mit6 allele (located at 13.7 cM from the centromere), together with a gain of the BALB/c allele of the more distal (29.6 cM) D15Mit64 marker, suggesting somatic recombination. LOH in the region close to c-myc was detected in a proportion of tumors with AI-15. The observation of complex genetic alterations includes somatic recombination, AI and LOH involving chromosome 15 in tumors induced by a myc transgene. This indicates that at least two genes in addition to c-myc on this chromosome can be involved in lymphoma development.

High levels of phosphorylated c-Jun, Fra-1, Fra-2 and ATF-2 proteins correlate with malignant phenotypes in the multistage mouse skin carcinogenesis model.

Analysis of the functions of AP-1 transcription factor in cellular systems has shown its key role as a mediator of oncogenic signals. The employment of suitable animal model systems greatly facilitates the study of changes in the composition and activity of the AP-1 complex. Here, we have analysed the quantitative and qualitative changes of AP-1 at different stages of carcinogenesis in mouse skin cell lines, derived from tumours induced by chemical mutagens. The findings of this study suggest that elevated AP-1 DNA binding and transactivation activity characterize the carcinoma cell lines, most notably the highly malignant spindle carcinomas. In addition, increased amounts and post-translational modifications of c-Jun, Fra-1, Fra-2 and ATF-2 proteins account for a high percentage of the increased AP-1 activity. Remarkably, high levels of phosphorylated ATF-2 protein were detected in malignant cell lines, indicating a novel role of ATF-2 in tumour progression. c-Jun and ATF-2 proteins are phosphorylated by highly active JNK kinases present in tumour cells. Finally, our results indicate distinct functions for different AP-1 components in the promotion and progression of mouse skin tumours. Oncogene (2000) 19, 4011 - 4021.

Targeting gene expression to tumor cells with loss of wild-type p53 function.

The tumor suppressor protein p53 is a transcription factor that can positively regulate the expression of critical target genes involved in negative control of cell growth or induction of apoptosis; p53 is also able to suppress the transcription of other genes by virtue of its ability to bind components of the basal transcription machinery. Over 50% of human tumors are characterized by p53 mutations that result in a loss of wild-type p53 (wtp53) function in the transcriptional control of these target genes. We have exploited this loss of p53 function in the regulation of gene transcription to develop a novel gene therapy strategy that maximizes expression of the potential therapeutic gene in tumors while simultaneously down-regulating the same gene in normal cells. In one construct (unit I), the potential therapeutic gene (in this case represented by a luciferase reporter) is placed under the control of a promoter such as the heat shock protein 70 gene promoter, which is repressed by wtp53 but overexpressed in many tumor cells with defective p53 function. Residual expression of the reporter in normal cells is repressed by cotransfection of another construct (unit II) consisting of a repressor of unit I under the control of a promoter that is activated by wtp53 expression. Unit II contains a promoter with a consensus wtp53 binding site driving a transcriptional repressor or an antisense construct for the gene in unit I. Our results suggest that this dual control approach may represent a strategy with wide applications in the field of cancer gene therapy.