When cancer meets quantum mechanics.

To date, classical deterministic Newtonian physics has been used by biologists to describe living processes. However, it is increasingly appreciated that the probabilistic view offered by quantum mechanics more accurately describes the behavior of atoms and materials in all systems. Here, we discuss how the concepts of quantum mechanics can be applied to biological processes involved in cancer. We present a concise summary inspired by Heisenberg's Uncertainty Principle to describe our «Genetic Environmental Field Hypothesis¼. Combining the uncertainties of genetic changes as expressed by epigenetic changes and/or somatic mutations with the uncertainties of environmental changes, cells may become cancerous as a way to increase entropy. Throughout the paper we will utilize the H19 gene system as an example. Using the concepts of quantum mechanics to describe oncological processes may provide novel directions in our understanding of cancer.

H19 non-coding RNA in urine cells detects urothelial carcinoma: a pilot study.

CONTEXT: Urothelial carcinoma (UC) is common and highly recurrent. Diagnosis and follow-up involve invasive cystoscopies. OBJECTIVE: To evaluate H19 RNA in urine cells as diagnostic tool for UC. MATERIALS AND METHODS: RT-PCR analysis of urine samples from healthy volunteers and UC patients. RESULTS: H19 RNA was unequivocally detected in the urine of 90.5% of patients and 25.9% of controls. H19 copies were three orders of magnitude higher in patients. Receiver operating characteristic analysis showed an area under the curve of 0.933. CONCLUSIONS: This pilot study shows that urinary cell H19 is a highly sensitive test for UC and pending verification could transform patient management.

The role of the oncofetal H19 lncRNA in tumor metastasis: orchestrating the EMT-MET decision.

Long non-coding RNA (lncRNA) genes are emerging as key players in the metastatic cascade. Current evidence indicate that H19 lncRNA and the microRNA(miRNA) miR-675, which is processed from it, play crucial roles in metastasis, through the regulation of critical events specifically the epithelial to mesenchymal (EMT) and the mesenchymal to epithelial transitions (MET). This review summarizes recent mechanistic pathways and tries to put together seemingly conflicting data from different reports under one proposed general scheme underlying the various roles of H19/miR-675 in the metastatic cascade. We propose several approaches to harnessing this knowledge for translational medicine.

The H19 Long non-coding RNA in cancer initiation, progression and metastasis - a proposed unifying theory.

The imprinted oncofetal long non-coding RNA (lncRNA) H19 is expressed in the embryo, down-regulated at birth and then reappears in tumors. Its role in tumor initiation and progression has long been a subject of controversy, although accumulating data suggest that H19 is one of the major genes in cancer. It is actively involved in all stages of tumorigenesis and is expressed in almost every human cancer. In this review we delineate the various functions of H19 during the different stages in the complex process of tumor progression. H19 up-regulation allows cells to enter a "selfish" survival mode in response to stress conditions, such as destabilization of the genome and hypoxia, by accelerating their proliferation rate and increasing overall cellular resistance to stress. This response is tightly correlated with nullification, dysfunction or significant down-regulation of the master tumor suppressor gene P53. The growing evidence of H19's involvement in both proliferation and differentiation processes, together with its involvement in epithelial to mesenchymal transition (EMT) and also mesenchymal to epithelial transition (MET), has led us to conclude that some of the recent disputes and discrepancies arising from current research findings can be resolved from a viewpoint supporting the oncogenic properties of H19. According to a holistic approach, the versatile, seemingly contradictory functions of H19 are essential to, and differentially harnessed by, the tumor cell depending on its context within the process of tumor progression.

The non-coding RNAs of the H19-IGF2 imprinted loci: a focus on biological roles and therapeutic potential in Lung Cancer.

Since it was first described, the imprinted cluster 11p15.5 has been reported to be deregulated in a variety of pediatric and adult cancers including that of the lung. Both protein coding and non-coding genes functioning as oncogenes or as tumor suppressor genes reside within this cluster. Oncomirs that can function as oncogenes or as tumor suppressors have also been reported. While a complete account of the role played by the 11p15.5 imprinted cluster in lung cancer is beyond the scope of this review, we will focus on the role of the non-coding RNAs processed from the H19-IGF2 loci. A special emphasis will be given to the H19/miR-675 gene locus. Their potential diagnostic and therapeutic use in lung cancer will be described.

Oncofetal H19 RNA promotes tumor metastasis.

The oncofetal H19 gene transcribes a long non-coding RNA(lncRNA) that is essential for tumor growth. Here we found that numerous established inducers of epithelial to mesenchymal transition(EMT) also induced H19/miR-675 expression. Both TGF-ß and hypoxia concomitantly induced H19 and miR-675 with the induction of EMT markers. We identified the PI3K/AKT pathway mediating the inductions of Slug, H19 RNA and miR-675 in response to TGF-ß treatment, while Slug induction depended on H19 RNA. In the EMT induced multidrug resistance model, H19 level was also induced. In a mouse breast cancer model, H19 expression was tightly correlated with metastatic potential. In patients, we detected high H19 expression in all common metastatic sites tested, regardless of tumor primary origin. H19 RNA suppressed the expression of E-cadherin protein. H19 up-regulated Slug expression concomitant with the suppression of E-cadherin protein through a mechanism that involved miR-675. Slug also up-regulated H19 expression and activated its promoter. Altogether, these results may support the existence of a positive feedback loop between Slug and H19/miR-675, that regulates E-cadherin expression. H19 RNA enhanced the invasive potential of cancer cells in vitro and enhanced tumor metastasis in vivo. Additionally, H19 knockdown attenuated the scattering and tumorigenic effects of HGF/SF. Our results present novel mechanistic insights into a critical role for H19 RNA in tumor progression and indicate a previously unknown link between H19/miR-675, Slug and E-cadherin in the regulation of cancer cell EMT programs.

NAF-1 and mitoNEET are central to human breast cancer proliferation by maintaining mitochondrial homeostasis and promoting tumor growth.

Mitochondria are emerging as important players in the transformation process of cells, maintaining the biosynthetic and energetic capacities of cancer cells and serving as one of the primary sites of apoptosis and autophagy regulation. Although several avenues of cancer therapy have focused on mitochondria, progress in developing mitochondria-targeting anticancer drugs nonetheless has been slow, owing to the limited number of known mitochondrial target proteins that link metabolism with autophagy or cell death. Recent studies have demonstrated that two members of the newly discovered family of NEET proteins, NAF-1 (CISD2) and mitoNEET (mNT; CISD1), could play such a role in cancer cells. NAF-1 was shown to be a key player in regulating autophagy, and mNT was proposed to mediate iron and reactive oxygen homeostasis in mitochondria. Here we show that the protein levels of NAF-1 and mNT are elevated in human epithelial breast cancer cells, and that suppressing the level of these proteins using shRNA results in significantly reduced cell proliferation and tumor growth, decreased mitochondrial performance, uncontrolled accumulation of iron and reactive oxygen in mitochondria, and activation of autophagy. Our findings highlight NEET proteins as promising mitochondrial targets for cancer therapy.

The increasing complexity of the oncofetal h19 gene locus: functional dissection and therapeutic intervention.

The field of the long non-coding RNA (lncRNA) is advancing rapidly. Currently, it is one of the most popular fields in the biological and medical sciences. It is becoming increasingly obvious that the majority of the human transcriptome has little or no-protein coding capacity. Historically, H19 was the first imprinted non-coding RNA (ncRNA) transcript identified, and the H19/IGF2 locus has served as a paradigm for the study of genomic imprinting since its discovery. In recent years, we have extensively investigated the expression of the H19 gene in a number of human cancers and explored the role of H19 RNA in tumor development. Here, we discuss recently published data from our group and others that provide further support for a central role of H19 RNA in the process of tumorigenesis. Furthermore, we focus on major transcriptional modulators of the H19 gene and discuss them in the context of the tumor-promoting activity of the H19 RNA. Based on the pivotal role of the H19 gene in human cancers, we have developed a DNA-based therapeutic approach for the treatment of cancers that have upregulated levels of H19 expression. This approach uses a diphtheria toxin A (DTA) protein expressed under the regulation of the H19 promoter to treat tumors with significant expression of H19 RNA. In this review, we discuss the treatment of four cancer indications in human subjects using this approach, which is currently under development. This represents perhaps one of the very few examples of an existing DNA-based therapy centered on an lncRNA system. Apart from cancer, H19 expression has been reported also in other conditions, syndromes and diseases, where deregulated imprinting at the H19 locus was obvious in some cases and will be summarized below. Moreover, the H19 locus proved to be much more complicated than initially thought. It houses a genomic sequence that can transcribe, yielding various transcriptional outputs, both in sense and antisense directions. The major transcriptional outputs of the H19 locus are presented here.

Use of preclinical models to assess the therapeutic potential of new drug candidates for bladder cancer.

The purpose of this review is to demonstrate a successful use of preclinical models of bladder cancer to confirm the therapeutic potential of new promising drug candidates. The bladder has long been thought to be an ideal target for investigating therapies. When developing a new antineoplastic pharmaceutical agent, the bladder should be considered for use as an experimental model demonstrating initial proof of concept that if successful can be later assessed in further cancer indications. Non-muscle-invasive bladder carcinoma can be removed by transurethral resection but these cancers tend to recur in most patients. Conventional treatments decrease the recurrence rate but are associated with side effects and frequent failures. Thus, there is an obvious need for the development of highly effective targeted therapies with limited side effects. Accordingly, a double-promoter vector was developed, expressing diphtheria toxin A (DTA) under control of two different regulatory promoter sequences, H19 and IGF2. This vector was then used to transfect and to eradicate tumor cells in bladder cancer models, effectively destroying tumor cells without affecting normal cells. Our studies demonstrate the potential efficacy of the therapeutic vector and should be a solid base for future clinical studies. These models illuminate the path for future investigations of new drug candidates for bladder cancer.

H19-promoter-targeted therapy combined with gemcitabine in the treatment of pancreatic cancer.

Pancreatic cancer is the eighth cancer leading cause of cancer-related death in the world and has a 5-year survival rate of 1-4% only. Gemcitabine is a first line agent for advanced pancreatic therapy; however, its efficacy is limited by its poor intracellular metabolism and chemoresistance. Studies have been conducted in an effort to improve gemcitabine treatment results by adding other chemotherapeutic agents, but none of them showed any significant advantage over gemcitabine monotherapy. We found that 85% of human pancreatic tumors analyzed by in situ hybridization analyses showed moderated to strong expression of the H19 gene. We designed a preclinical study combining gemcitabine treatment and a DNA-based therapy for pancreatic cancer using a non viral vector BC-819 (also known as DTA-H19), expressing the diphtheria toxin A chain under the control of the H19 gene regulatory sequences. The experiments conducted either in an orthotopic and heterotopic pancreatic carcinoma animal model showed better antitumor activity following the sequential administration of the vector BC-819 and gemcitabine as compared to the effect of each of them alone. The results presented in the current study indicate that treatment with BC-819 in combination with gemcitabine might be a viable new therapeutic option for patients with advanced pancreatic cancer.

Transcriptional targeting of glioblastoma by diphtheria toxin-A driven by both H19 and IGF2-P4 promoters.

BACKGROUND: The H19-IGF2 locus is either highly expressed and/or shows aberrant allelic pattern of expression in a large array of human cancers, while rarely expressed in the corresponding normal tissue. Preclinical, clinical studies and human compassionate using a DNA plasmid containing H19 and/or IGF2-P4 regulatory sequences that drive the expression of an intracellular toxin [diphtheria toxin A-fragment (DTA)] have demonstrated promising results in several types of carcinomas. Recently we reported that a single construct that expresses DTA under the control of both H19 and IGF2 P4 promoters showed superior efficacy in vitro as well as in vivo, in comparison to a single promoter construct in bladder carcinoma. Here we extended this approach to glioblastoma and tested the antitumor efficacy of the double promoter DTA-expressing vector (H19-DTA-P4-DTA) in vitro as well as in heterotopic animal model. H19 gene expression was tested by in-situ hybridization (ISH) and by quantitative Real-Time PCR (qRT-PCR) in samples of diffuse glioma. METHODS: IGF2-P4 gene expression was tested by qRT-PCR as well. RESULTS: Both H19 and IGF2-P4 transcripts were highly expressed in high grade gliomas. Furthermore, significant H19 expression in other types of primary brain tumors as well as in brain metastases was detected by ISH. Both A172 and U87 human glioblastoma cell lines showed high expression of IGF2-P4 while the A172 cell line showed high expression of H19 RNA as well. H19-DTA-P4-DTA exhibited superior cytotoxic activity compared to the single promoter expression vectors, in U87 and A172 glioblastoma cell lines in vitro and showed antitumoral efficacy in heterotopic glioblastoma animal model. CONCLUSIONS: Our findings indicate antitumoral efficacy against glioblastoma of the targeted double promoter vector H19-DTA-P4-DTA, both in-vitro and in-vivo. Thus, its test in orthotopic animal model of glioblastoma as well as in clinical trials is warranted.

Regional therapy with DTA-H19 vector suppresses growth of colon adenocarcinoma metastases in the rat liver.

Curative surgery is possible in barely 10% of patients with colorectal liver metastases and combined treatment modalities scarcely improve survival in this group of patients. Hence, investigations of new therapeutic modalities are crucial. Overexpression of the H19 gene in liver metastases points to H19 as a target for cancer gene therapy. Here we have evaluated the possibility of regional intra-arterial treatment of liver meta-stases with the DTA-H19 plasmid. Intra-arterial treatment of a total dose of 2.5 mg (repeated injections of 500 µg DTA-H19 plasmid each dose after the first injection of 1000 µg) caused a significant delay in the tumor growth compared to control group. All of the tumors treated with the control vector increased in size, whereas 35.7% of the tumors in the groups treated with a total amount of 2.5 mg DTA-H19 plasmid shrank in size. The present study showed that the DTA-H19 plasmid administered intra-arterially significantly delayed the tumor growth and even resulted in tumor regression in high percentage of the treated animals with liver metastases of colon cancer. Since human liver metastases demonstrated overexpression of the H19 gene, regional administration of the plasmid seems to be a promising therapeutic approach.

Targeting diphtheria toxin and TNF alpha expression in ovarian tumors using the H19 regulatory sequences.

BACKGROUND: There are currently no effective therapies for the treatment of ovarian cancer ascites fluid (OCAF). H19 is an RNA oncofetal gene that is present at high levels in human cancer tissues (ovarian cancer and OCAF among them), while existing at a nearly undetectable level in the surrounding normal tissue. There is evidence for a synergistic effect in cell cytotoxicity mediated by TNFα and diphtheria toxin in sensitive and resistant human ovarian tumor cell line. Thus, we tested the cytotoxic effect of TNF-α cytokine, together with the diphtheria toxin, in the therapy of ovarian cancer. METHODS: The therapeutic potential of toxin vectors carrying the DT-A gene alone (pH19-DTA), or in combination with the TNF-α gene (pH19-TNF-DTA), driven by H19 regulatory sequences were tested in ovarian carcinoma cell lines and in a heterotopic ovarian cancer model. RESULTS: The toxin vectors showed a high killing capacity when transfected into different ovarian cancer cell lines. In addition, intratumoral injection of the toxin vector into ectopically developed tumors caused 40% inhibition of tumor growth. The killing effect after injection of pH19-TNF-DTA plasmid into ectopically developed tumors was significantly higher than that showed by the pH19-DTA plasmid alone, particularly in diphtheria toxin and TNF resistant tumors. CONCLUSIONS: These observations may be the first step towards a major breakthrough in the treatment of human ovarian cancer. It should enable us to identify likely non-responders in advance, and to treat patients who are resistant to all known therapies, thereby avoiding treatment failure coupled with unnecessary suffering and cost.

Treatment of ovarian cancer ascites by intra-peritoneal injection of diphtheria toxin A chain-H19 vector: a case report.

INTRODUCTION: Ovarian cancer ascitic fluid, which contains malignant cells, is usually present in women with an advanced stage disease. There are currently no effective therapies for the treatment of ovarian cancer ascitic fluid. We developed a new therapeutic strategy to target expression of the diphtheria toxin fragment A gene in ovarian tumor cells under the control of H19 regulatory sequences. CASE PRESENTATION: A 64-year-old Caucasian woman was diagnosed with a stage IIIc epithelial ovarian cancer. She suffered from progressive disease, accumulation of malignant ascites that needed to be drained weekly, abdominal pain, vomiting, anorexia and severe weakness. Infusion of the diphtheria toxin A chain-H19 plasmid into the peritoneum of our patient resulted in complete resolution of the ascites with minimum adverse events. CONCLUSION: On the basis of this preliminary experience, we are currently conducting an extensive Phase I study on a larger number of patients in order to assess the safety and preliminary efficacy of this novel patient-oriented treatment approach.

The oncofetal H19 RNA connection: hypoxia, p53 and cancer.

Expression of the imprinted H19 gene is remarkably elevated in a large number of human cancers. Recently, we reported that H19 RNA is up-regulated in hypoxic stress and furthermore, it possesses oncogenic properties. However, the underlying mechanism(s) of these phenomena remain(s) unknown. Here we demonstrate a tight correlation between H19 RNA elevation by hypoxia and the status of the p53 tumor suppressor. Wild type p53 (p53(wt)) prevents the induction of H19 upon hypoxia, and upon its reconstitution in p53(null) cells. The last case is accompanied by a decrease in cell viability. The p53 effect is nuclear and seems independent of its tetramerization. Furthermore, using knockdown and over-expression approaches we identified HIF1-alpha as a critical factor that is responsible for H19 induction upon hypoxia. Knocking down HIF1-alpha abolishes H19 RNA induction, while its over-expression significantly enhances the H19 elevation in p53(null) hypoxic cells. In p53(wt) hypoxic cells simultaneous suppression of p53 and over-expression of HIF1-alpha are needed to induce H19 significantly, while each treatment separately resulting in a mild induction, indicating that the molecular mechanism of p53 suppression effect on H19 may at least in part involve interfering with HIF1-alpha activity. In vivo a significant increase in H19 expression occurred in tumors derived from p53(null) cells but not in p53(wt) cells. Taken together, our results indicate that a functional link exists between p53, HIF1-alpha and H19 that determines H19 elevation in hypoxic cancer cells. We suggest that this linkage plays a role in tumor development.

Development of targeted therapy for ovarian cancer mediated by a plasmid expressing diphtheria toxin under the control of H19 regulatory sequences.

BACKGROUND: Ovarian cancer ascites fluid (OCAF), contains malignant cells, is usually present in women with an advanced stage disease and currently has no effective therapy. Hence, we developed a new therapy strategy to target the expression of diphtheria toxin gene under the control of H19 regulatory sequences in ovarian tumor cells. H19 RNA is present at high levels in human cancer tissues (including ovarian cancer), while existing at a nearly undetectable level in the surrounding normal tissue. METHODS: H19 gene expression was tested in cells from OCAF by the in-situ hybridization technique (ISH) using an H19 RNA probe. The therapeutic potential of the toxin vector DTA-H19 was tested in ovarian carcinoma cell lines and in a heterotopic animal model for ovarian cancer. RESULTS: H19 RNA was detected in 90% of patients with OCAF as determined by ISH. Intratumoral injection of DTA-H19 into ectopically developed tumors caused 40% inhibition of tumor growth. CONCLUSION: These observations may be the first step towards a major breakthrough in the treatment of human OCAF, while the effect in solid tumors required further investigation. It should enable us to identify likely non-responders in advance, and to treat patients who are resistant to all known therapies, thereby avoiding treatment failure.

Highly upregulated in liver cancer noncoding RNA is overexpressed in hepatic colorectal metastasis.

BACKGROUND: The highly upregulated in liver cancer (HULC) gene transcribes to an mRNA-like noncoding RNA (ncRNA) by the RNA polymerase II and processed by capping, splicing and polyadenylation. It is specifically expressed in the hepatocytes with striking upregulation in hepatocellular carcinoma (HCC). OBJECTIVES: To study the expression levels of HULC in normal colorectal samples, primary colorectal carcinomas and in secondary tumors formed from colorectal carcinomas that metastasize to either the liver or the lymph nodes, taken from the same patients. Also a panel of carcinoma cell lines is tested for HULC expression. BASIC METHODS: Semiquantitative reverse transcriptase-PCR technique is used to detect for HULC expression in study specimens and cell lines. RESULTS: Consistent with the previous report, HULC is neither expressed in primary colorectal carcinomas samples nor in their normal counterparts. We show for the first time those colorectal carcinomas that metastasize to the livers but not to lymph nodes experience an upregulation of HULC ncRNA in all the samples tested (n= 8), with a strong-to-moderate expression in six out of eight. Moreover HULC is not expressed in the majority of carcinoma cell lines tested and also in samples of normal bladder and bladder cancers of various grades. We also show that HULC ncRNA is upregulated in two hepatocellular carcinoma cell lines producing HBV relevant to their parental lines that do not produce HBV. CONCLUSION: Our results presented here indicate that HULC expression is not confined to HCC, but also to those colorectal carcinomas that metastasize to the liver.

The H19 non-coding RNA is essential for human tumor growth.

BACKGROUND: Mutations and epigenetic aberrant signaling of growth factors pathways contribute to carcinogenesis. Recent studies reveal that non-coding RNAs are controllers of gene expression. H19 is an imprinted gene that demonstrates maternal monoallelic expression without a protein product; although its expression is shut off in most tissues postnatally, it is re-activated during adult tissue regeneration and tumorigenesis. Moreover, H19 is highly expressed in liver metastasis derived from a range of carcinomas. The objective of this study is to explore the role of H19 in carcinogenesis, and to determine its identification as an anti-tumor target. METHODOLOGY/PRINCIPLE FINDINGS: By controlling oxygen pressure during tumor cell growth and H19 expression levels, we investigated the role of H19 expression in vitro and in vivo in hepatocellular (HCC) and bladder carcinoma. Hypoxia upregulates the level of H19 RNA. Ablations of tumorigenicity of HCC and bladder carcinomas in vivo are seen by H19 knockdown which also significantly abrogates anchorage-independent growth after hypoxia recovery, while ectopic H19 expression enhances tumorigenic potential of carcinoma cells in vivo. Knocking-down H19 message in hypoxic stress severely diminishes p57(kip2) induction. We identified a number of potential downstream targets of H19 RNA, including angiogenin and FGF18. CONCLUSIONS: H19 RNA harbors pro-tumorigenic properties, thus the H19 gene behaves as an oncogene and may serve as a potential new target for anti-tumor therapy.

Differential expression of kinase genes in primary hyperparathyroidism: adenoma versus normal and hyperplastic parathyroid tissue.

CONTEXT: Differentiation between adenoma and hyperplasia or even normal parathyroid tissue is difficult and based mainly on the surgeon's skill. Exploration of genes that express differentially in these various tissues using microarrays and other sophisticated research tools will enable identification and perhaps development of new methods of perioperative diagnosis. OBJECTIVE: To assemble a panel of kinase genes to differentiate parathyroid adenoma from normal and hyperplastic parathyroid tissue. DESIGN: RNA was extracted from adenoma, hyperplasia, and normal parathyroid tissue and hybridized to a microarray containing 359 human cDNAs of known kinase genes. Signals of exposure were scanned and quantified with software for digital image analysis. Semiquantitative reverse transcriptase polymerase chain reaction analysis of sample genes was performed, up-regulated or down-regulated, to validate the microarray results. RESULTS: The ratio values considered significant (<0.5 or >1.5) suggest that genes up-regulated in parathyroid adenoma are those responsible for blood vessel angiogenesis and genes belonging to the cyclin-dependent kinase inhibitor groups. Genes down-regulated in parathyroid adenoma are related to cellular growth and apoptosis--genes from the mitogen-activated protein kinase group and DNA-dependent protein kinase group. An interesting gene down-regulated in the parathyroid adenoma samples is related to the serine/threonine protein kinases that exert a key function in calcium handling. A panel of 5 genes was defined: p19, p21 and the gene for vascular endothelial growth factor from the up-regulated group, and the gene for protein kinase C and SGK from the down-regulated group. Reverse transcriptase polymerase chain reaction confirmed the microarray results for these genes. CONCLUSIONS: The kinase genes panel presented can be used to differentiate parathyroid adenoma from normal and hyperplastic parathyroid tissue in particular when histopathology fails to provide a decisive diagnosis.

Regulatory sequences of H19 and IGF2 genes in DNA-based therapy of colorectal rat liver metastases.

BACKGROUND: Malignant tumors of the liver are among the most common causes of cancer-related death throughout the world. Current therapeutic approaches fail to control the disease in most cases. This study seeks to explore the potential utility of transcriptional regulatory sequences of the H19 and insulin growth factor 2 (IGF2) genes for directing tumor-selective expression of a toxin gene (A fragment of diphtheria toxin), delivered by non-viral vectors. METHODS: The therapeutic potential of the toxin vectors driven by the H19 and the IGF2-P3 regulatory sequences was tested in a metastatic model of rat CC531 colon carcinoma in liver. RESULTS: Intratumoral injection of these vectors into colon tumors implanted in the liver of rats induced an 88% and a 50% decrease respectively in the median tumor volume as compared with the control groups. This therapeutic action was accompanied by increased necrosis of the tumor. Importantly, no signs of toxicity were detected in healthy animals after their treatment by the toxin expression vectors. CONCLUSIONS: DT-A was preferentially expressed in liver metastases after being transfected with H19 or IGF2-P3 promoter-driven DT-A expression plasmids, causing a very significant inhibition of tumor growth as a result of its cytotoxic effect. Our findings strongly support the feasibility of our proposed therapeutic strategy, which may contribute to open new gene therapeutic options for human liver metastases.