I. Introduction

Throughout time, the human race has striven for immortality. Testimonials are the creation of physical and mental monuments, such as pyramids, or E=mc².A concept of life after death is a component of both Eastern and Western religions. In fact, immortality has been achieved: the simplest form of life, the cell, regularly achieves immortality. By transforming itself into a cancer cell, it will live on and on. However, its immortality may be deadly to higher organisms.

Cancer has been a devastating disease throughout time. In 2004, more than half a million Americans died of cancer – more than all those who fell in America’s wars throughout history.

Although investments in research and drug developments soared in the last three decades, the mortality rate from cancer is still high. 30 years ago, 50% of cancer patients survived 5 years after diagnosis. Since then, minor progress increased that rate to 63%. This rise occurred due to change in personal habits, e.g. less smoking, and earlier detection of tumors.

Still, one out of two men and one out of three women may suffer from tumors during their life. This is a horrifying prediction which can be compared to a scenario where everyday two twin towers collapse.

These data explain why people must devote resources to research. Cancer research has generated a rich and complex body of knowledge.

Cancer now is known to involve dynamic changes in the genome. The foundation has been set for the discovery of mutations that produce oncogenes with dominant gain of function and tumor suppressor genes with recessive loss of function (Bishop and Weinberg, 1996).

Several lines of evidence indicate that tumorogenesis in humans is a multi-step process. These steps reflect genetic alteration that drives the progressive transformation of normal human cells into highly malignant derivatives. The vast catalog of cancer cell genotypes is a manifestation of six essential alterations in cell physiology that collectively dictate malignant growth: self-sufficing-in-growth signals, insensitivity to growth-inhibitory signals, evasion of programmed cell death (apoptosis), limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis (Hanahan and Weinberg, 2000).

The genesis of cancer teaches us the complete lesson of evolution. In general, the pace of evolution, whether in malignant cells or a population of organisms, is dependent upon four factors: rate of mutation, rate of reproduction, number of individuals/cells and selective advantage gained by successful mutants (Glassman and Hochberg, 1997).

Genetic instability is at the heart of malignancy, characterized by a high rate of mutations. This mutational cascade is shaped by natural selection to gain cellular autonomy. The results are a high rate of proliferation, invasion and conquest of a new habitat.

Metastasis is the fatal event for patients and is particular evidence of a strong process of selection. To disseminate, tumor cells must loosen their adhesion to their neighbors, move through and escape their original tissue, cross through a vassal lamina and endothelium to reach the interior of a blood or lymphatic vessel, make an exit from the circulation and then survive in a new environment.

Over billions of years of evolution, pressures remained to favor rapidly dividing cells. Thus the step between germ and somatic cells was a precipitous one. Somatic cells forsook their ability to vertically reproduce, in order to specialize. This enabled them to enhance circumstances for the germ cells, which would be responsible for the next generation.

Genetic programs curbed the reproduction of the somatic cells. The loss of these control programs (tumor suppressor genes) and activation of proliferation (prot-oncogenes) are the essence of oncogenesis.

In the late 1960s, several investigators suggested that the first genetic molecule on earth was RNA. They proposed the establishment of what is now called the RNA world – a world in which RNA catalyzed all the reactions necessary for a precursor of life’s last common ancestor to survive and replicate.

During the last 10 years, a fair amount of evidence has lent credence to the idea that the hypothetical RNA world did exist and led to the advent of life based on DNA, RNA and protein. In 1983, Thomas R. Cech and Sidney Altman independently discovered the first known ribozymes, enzymes made of RNA. So far no RNA molecules that direct the replication of other RNA molecules have been identified.

H19 was the first oncofetal RNA discovered. In this review, we will present the role of H19 in humans, its proposed mechanism of action and its use as a therapy against cancer.

Finally, we would like to define the term therapognostic. We know now that cancer is a very complicated disease. The hallmark of cancer cells; mutability, natural selection, and change in biotope, bring about that the primary neoplasm is different from the metastatic one. Even more, each neogrowth in one patient might be different from the other. We have to adopt treatment tailored to the gene expression in each tissue of the patient. We have to develop a method where the diagnostic will be prerequisite of the treatment. We define this strategy therapognostic matching therapy and diagnosis.

II. Imprinting and regulation of the H19 gene

A. H19 is an imprinted gene with no protein product

In some animals including mammals, a number of genes have been found to be subject of genomic imprinting, a phenomenon of monoallelic expression depending on the parent of origin. The parental alleles of imprinted genes are marked during gametogenesis before fertilization, such that they are transcriptionally silenced at one of the parental alleles in the offspring, resulting in differential gene expression in a tissue specific way

H19 was the first human imprinted non-coding gene to be identified showing expression of only the maternal allele (Rachmilewitz et al, 1992 B; Zhang and Tycko, 1992). It is also imprinted in mice (Bartolomei et al, 1991). Parentally methylated regions in germ line are present upstream of the H19 promoter. When a mutation in the methylation region of the maternal H19 gene locus occurs, the methylation imprinting pattern is identical to paternal wild type causing silencing of the H19 gene. On the other hand, loss of paternal-dependent silencing causes over-expression of the H19 gene (Cui et al, 2002).

H19 was mapped on the short arm of chromosome 11, band 15.5, homologous to a region of murine chromosome 7 (Leibovitch et al, 1991). Imprinted genes including H19 are usually found organized as clusters harboring long range, cis-acting DNA sequences known as imprint control elements. Both physical and functional locations of H19 gene within the cluster are conserved between human and mice.

110-200Kb centromeric to H19 located insulin-like growth factor 2 (IGF2) gene (Zemel et al, 1992). IGF2 and H19 are reciprocally imprinted and have been found to show reciprocal regulation in a number of tissues. The opposite pattern of imprinting and expression of H19 and IGF2 was initially explained by assuming a so called “enhancer competition model” (Zemel et al, 1992; Bartolomie et al, 1993).

According to this model both H19 and IGF2 compete for a common set of enhancers. The maternal chromosome of H19 gene monopolizes the shared enhancers by virtue of its proximity and/or greater promoter strength. On the paternal chromosome, allele specific methylation of H19 would silence the gene and allow IGF2 access to the enhancers.

However it was also shown that enhancer competition between H19 and IGF2 is not sufficient to explain their imprinting status (Schmidt et al, 1999). Another model called the boundary model was proposed (Bell and Felsenfeld, 2000).

Transcription of these genes is controlled by a set of shared enhancers downstream of H19, and a differentially methylated domain (DMD) upstream of H19. The DMD can bind to a protein called (CTCF) through a binding site, and this protein can act as chromatin insulator.

On the maternally inherited chromosome, CTCF binds to unmethylated DMD creating a chromatin insulator which prevents the IGF2 promoter from gaining access to downstream enhancers. On a paternally inherited allele, CTCF can't bind because DMD is methylated. This prevents the action of the insulator allowing the promoter of the paternal IGF2 allele to interact with the downstream enhancers (Bell and Felsenfeld, 2000; Hark et al, 2000). A detailed description of genomic imprinting of H19 and IGf2 genes lies beyond the scope of this review. The reader can refer to (Arney, 2003; Verona et al, 2003).

Non-coding RNAs have increasingly been found associated with imprinted genes. H19 belongs to a group of genes that very likely does not code for a protein product (Brannan et al, 1990). Although H19 RNA undergoes all the classical properties of coding transcripts--it is transcribed by RNA-polymerase II, capped, spliced, polyadenylated and transported to the cytoplasm--it seems to function as an RNA and not as a protein.

H19 was found associated with polyribosomes in different human cells indicating that it probably is a ribo-regulator, an RNA molecule with regulatory functions, such as (3' untranslated region of tropomysin mRNA for muscle cell differentiation and Xist for X-inactivation (Erdmann et al, 2001). The genes isolated from humans and mice consist of five exons separated by unusually short introns (80-96 nucleotides in humans).

Both human and mice H19 RNAs have multiple open reading frames (ORFs), the largest of which can potentially code for 256 and 132 amino acid-long peptides. However, there is no similarity between the two amino acid sequences, although comparison between the human and mouse H19 gene revealed an overall 77% sequence identity (Brannan et al, 1990).

The longest ORF of the human gene is preceded by four very short ORFs, which may be the reason for the complete repression of the main ORF translation in a cell-free translation system. Introduction of deletions and/or point mutations into the 5’untranslated region of an ectopic H19 gene, upstream of the largest ORF, enabled the production of the 26Kd protein, although this has not been detected in cells expressing an endogenous H19 gene (Joubel et al, 1996).

We recently reported the identification of an alternative splice variant of H19 RNA that lacks part of exon 1. This variant was detected in human embryonic and placental tissues, but not in bladder or hepatocellular carcinomas. A very low level of this variant was also detected in colon carcinoma. The observed pattern of expression suggests that this splice variant is a developmentally regulated H19 gene transcript (Matouk et al, 2004).

H19 gene was isolated by differential cDNA cloning from murine embryonic stem cells, which differentiate in vitro to embyoid bodies and as one of the genes (referred to as MyoH) involved in differentiation of myoblasts (Davis et al, 1987; Poirier et al, 1991).

H19 was initially isolated as a raf-regulating gene, involved in expression of a-fetoprotein in the mouse liver (Pachnis et al, 1988). Its human counterpart was identified based on homology with the murine gene. We have cloned and isolated H19 by differential screening of cDNA libraries of in vitro cultured differentiated cytotrophoblasts (Rachmilevitz et al, 1992 A).

B. H19 upstream effectors and regulation

Little is known today about the regulation of H19 gene expression. It was reported that H19 over-expression in breast adenocarcinoma was significantly correlated with the presence of steroid hormone receptors (Adriaenssens et al, 1998). Steroid hormones modulate H19 expression in the mammary glands and the uterus where H19 is up regulated by 17-b-estradoil and down regulated by progesterone (Adriaenssens et al, 1999).

Very recently, it was shown that H19 expression was regulated by a peptidic and a male steroid hormone (Berteaux et al, 2004). H19 gene expression is up regulated by prolactin, but dihydrotestosterone counteracted prolactin mediated enhancement, and this effect is observed in human androgen-dependent cancer cells, but not in human androgen-independent prostate cancer cells. The up regulation of H19 by prolactin occurs by the JAK2-STAT5 transduction pathway. Moreover, the H19 TATA-less promoter is efficiently repressed by wild type p53, which may place the H19 gene within the core mechanism of cell cycle control and cell proliferation, especially at the transition from the G1 to S phase (Dugimont et al, 1998). H19 gene expression is also activated during adult liver regeneration (Pachnis et al, 1984), after liver hepatoctomy (Yamamoto et al, 2004), during the luteal phase of the female menstrual cycle (Ariel et al, 1997b) and in wound healing (Kim et al, 1994).

Certain known carcinogens up regulate the expression of the H19 gene. In a study aimed to identify changes in gene expression patterns in the airway epithelium of disease-free smokers compared with a matched group of nonsmokers, a dramatic elevation of H19 RNA levels was detected in the airway epithelium of smokers without loss of imprinting (LOI) (Kaplan et al, 2003). BBN (a known carcinogen of the bladder also induces the expression of H19 gene in the rat model of bladder cancer (Elkin at al, 1998; Ariel et al, 2004). Moreover, Diethylnitrosamine (a known carcinogen of the liver) induces the expression of H19 in a mice model of hepatocellular carcinoma (Graveel et al, 2001).

It was also reported that H19 gene expression is up regulated in vitro in differentiating cells. It was shown that expression of the H19 gene is dependent on the differentiation stage (Davis et al, 1987; Pachnis et al, 1988; Poirier et al, 1991; Rachmilewitz et al, 1992a; Kopf et al, 1998).

In particular, we have shown that NT2 and NCCIT cells (derived from a human testicular germ cell tumor), do not express H19 gene. Retinoic acid, which plays an important role in the establishment of the differentiation pattern during embryogenesis, induces H19 expression in these cells, indicating the possibility that H19 RNA could mediate at least partly, the effect of retinoic acid on the differentiation of various tissues during embyogenesis (Kopf et al, 1998).

It was also found that H19 expression level is positively correlated with the differentiation stage of placental cytotrophoblasts both in vivo and in vitro (Rachmilewitz et al, 1992a). Leibovitch et al, (1995) also reported that the over-expression of the c-mos protein increases H19 RNA expression in the C2C12 muscle cell line, suggesting an interrelationship between these two gene products during muscle differentiation.

Additionally, no expression of H19 could be found in murine fertilized oocytes, the morula and early blastocyst. In the late blastocyst, however, H19 was found to be expressed in the trophectoderm (Poirier et al, 1991). H19 is therefore one of the earliest genes expressed during the first stages of embryonic differentiation.

A number of growth factors and cytokines were also reported to modulate the expression level of H19 RNA. Insulin, insulin-like growth factors (IGFI and II), epidermal growth factor, hepatocyte growth factor, tumor necrosis factor (TNF)-a, transforming growth factor (TGF)-b1, interferon (IFN)-g, and activators and inhibitors of protein kinase A and C, all were reported to modulate H19 gene expression in different cell lines, vascular smooth muscle cells, fetal adrenal cells and cultured adrenal cells (Han and Liau, 1992; Han et al, 1996) (Figure 1).

H19 gene was also shown to be regulated at the post-transcriptional levels. H19 gene is up regulated exclusively by stabilization of its RNA during muscle cell differentiation (Milligan et al, 2000). Turnover of primary transcripts is a major step in the regulation of mouse H19 gene expression (Milligan et al, 2002). Moreover, a link between IGF2 and H19 genes at post-transcriptional events during mammalian development has been suggested due to the ability of H19 RNA to bind four molecules of IGF2 mRNA binding proteins. This binding was sufficient to direct subcytoplasmic localization of H19 RNA to lamellipodia and perinuclear regions (Runge et al, 2000).

III. H19 and tumorigenesis

The imprinted cluster on the chromosome 11p15.5 has been implicated in a variety of disorders and cancer predisposition of both pediatric and adult tumors. This initially places the H19 gene as a candidate gene that fulfills a role in tumorgenesis. Following intensive investigation, contradictory roles have been proposed; a tumor suppressive role and an oncofetal role. In this section, we will present both proposals.

A. Tumor suppressor activity

Genetic analysis of the chromosome 11p15.5 region revealed a frequent correlation between the loss of the maternally inherited 11p15.5 region and many cases of Wilms’ tumor and some embryonal rhabdomyosarcomas (Scrable et al, 1989; Moulten et al, 1994). Loss of imprinting of IGF2 and inactivation of H19 has been implicated in the pathogenesis of embryonal tumors and Beckwith-Wiedmann syndrome (Ogawa et al 1993; Steenman et al, 1994). These correlations and other experimental findings can be explained by assuming the existence of a paternally imprinted tumor suppressor gene in this chromosomal area (Rainier et al, 1993).

In order to provide evidences for the proposal that H19 functions as a tumor suppressor gene, (Hao et al, 1993) introduced a construct expressing the H19 gene under the control of the metallothionein promoter into cells derived from a kidney tumor. Under the condition of high H19 expression, the cells, which showed prominent morphological changes, grew at a slower rate, had a much lower anchorage-independent growth rate in soft agar, and did not develop tumors when injected into nude mice, as did cells prior to their transfection with the H19 expression construct.

Additional support for the proposed tumor suppressor role for H19 came from the study of the role of the H19 gene in Syrian hamster embryo cell tumorigenicity (Isfort et al, 1997). In this report, the author presented evidence that alternation in H19 gene expression may play an important role in the Syrian hamster embryo cell transformation process. 75% of morphologically transformed cells, as compared with non-transformed cells, have reduced expression of the H19 gene. Moreover, re-establishment of wild-type H19 expression in tumorigenic Syrian hamster embryo cells suppressed the tumorigenicity of the transformed cells, indicating that the reduction in H19 expression observed at the morphological transformation stage may be important in cell tumorigenicity (Isfort et al, 1997).

Still more support for the possible tumor suppressor role of H19, has been postulated in the light of its possible role in controlling cellular differentiation. Thus by controlling differentiation H19 gene expression could result in differentiation of tumorigenic cells, thereby limiting their growth potential and tumorigenicity (Poirier et al, 1991; Davis et al, 1987; Pachnis et al, 1988; Kopf et al, 1998; Rachmilewitz et al, 1992 A). Moreover, the high frequency of inactivation of the H19 gene was found due to maternal allelic loss, by hyper-methylation of its promoter in sporadic hepatoblastoma (Fukuzawa et al, 1999), and in adrenocortical tumors (Goa et al, 2002).

B. H19 and human malignancies (Is H19 a tumor suppressor gene?)

However, accumulating data do not support the idea of H19 being a tumor suppressor gene. (Reid et al, 1996) and using the same cell lines as (Hao et al, 1993) who proposed the tumor suppressor activity of H19, found that H19 expression itself is not correlated with suppression of malignancy. Earlier data had already proposed that another locus on the short arm of chromosome 11 might be involved like p57^Kip2.

Moreover, studies of various tumors have demonstrated a re-expression or an over-expression of the H19 gene when compared to healthy tissue. In cancers of different etiologies and lineages, aberrant expression in allelic pattern was observed in some cases, suggesting that H19 may play a role in tumorigenesis. While H19 shows mono-allelic expression in most tissues throughout development, with the exception of germ cells at certain stages of maturation, and in extra-villous trophoblasts (Adam et al, 1996), bi-allelic expression of this gene, referred as “relaxation of imprinting” or LOI, have been found in an increasing number of cancers. H19 expression was demonstrated for example in a complete hydatiform mole, placental tissue that carries the exclusively paternal genome and gives rise to a malignant trophoblastic tumor choriocarcinoma (Ariel et al, 1994).

The kinds of tumors expressing H19, clearly demonstrated that LOI of H19 is not restricted to embryonal tumors. Moreover, some Wilms’ tumors and all neuroblastoma (embryonal tumors), retain mono-allelic expression of H19 (Wada et al, 1995). Bi-allelic expression of H19 was found for example in hepatocellular carcinoma (Kim et al, 1997), in liver neoplasms of albumin SV40 T antigen- transgenic rats (Manoharan et al, 2003), in lung adenocarcinoma (Kondo et al, 1995), in esophageal (Hibi et al, 1996), ovarian (Kim et al, 1998; Chen et al, 2000), rhabdomyosarcoma , cervical ( Douc-Rasy et al, 1996), bladder (Elkin et al, 1995), head and neck squamous cell carcinoma (el-naggar et al, 1999), colorectal (Cui et al, 2002), uterus (Hashimoto et al, 1997) and in testicular germ cell tumors (van Gurp et al, 1994;Verkerk et al, 1996).

Further, LOI may or may not associate with over-expression. This indicates that LOI leading to bi-allelic expression of H19 seems not to be crucial in the pathogenesis of cancers. The significance of LOI to tumorigenesis is not fully understood. Moreover, it was shown that H19 over-expression of ectopic origin conferred a proliferative advantage for breast epithelial cells in a soft agar assay and in several combined immunodeficient (SCID) mice (Lottin et al, 2002a).

In tumors formed by the injection of cells of a choriocarcinoma-derived cell line (JEG-3), and a bladder carcinoma cell line (T24P), the H19 level is very high when compared to the level of H19 in cells before injection (Elkin et al, 1995; Rachmilewitz et al, 1995; Lustig-Yariv et al, 1997). All of these observations and others contradict the proposal that H19 is a tumor suppressor gene.

Herein, we provide a list of cancers (which is still growing), in which H19 gene expression was detected:

A- Pediatric solid tumors

1. Wilms’ tumor.

2. Hepatoblastoma.

3. Embryonal rhabdomyosarcoma.

B- Germ cell tumors and trophoblastic tumors

1. Testicular germ cells tumors.

2. Immature teratoma of ovary.

3. Sacrococcygeal tumors.

4. Choriocarcinoma.

5. Placental site trophoblastic tumors.

C- Epithelial adult tumors

1. Bladder carcinoma.

2. Hepatocellular carcinoma.

3. Ovarian carcinoma.

4. Cervical carcinoma.

5. Lung carcinoma.

6. Breast carcinoma.

7. Squamous cell carcinoma in head and neck.

8. Esophageal carcinoma.

D- Neurogenic tumors

1. Astrocytoma.

2. Ganglioblastoma.

3. Neuroblastoma.

IV. The H19 gene in diagnosis, prognosis and DNA-based therapy

A. H19 as an Oncofetal RNA with both prognostic and diagnostic values

It is now widely recognized that developmental processes in general and embryogenesis in particular, share many biologic and morphologic features with neoplasms. These include characteristics linked to reduced differentiation, rapid proliferation rate, and the ability to invade. It has become apparent that many of the genes that are expressed during embryogenesis, and down regulated with tissue maturation, are re-expressed in cancer. These include, for example carcinoembryonic antigen, a fetoprotein and the b subunit of chorionic gonadotropin. These genes have been designated as oncofetal or onco-developmental genes.

The imprinted genes comprise a special group among embryonal genes. Imprinted genes play a pivotal role in embryogenesis and fetal growth and development (Tilghman, 1999; Ariel et al, 2000a).

The H19 transcript is an abundant RNA in the developing mouse embryo, in human embryonic tissues and in human placenta, and is down regulated postnatal in most tissues (Pachnis et al, 1984; Lustig et al, 1994). Our group has shown that H19 RNA is re-expressed in tumors arising from tissues which express it in fetal life. This expression is linked to the stage of differentiation and led to the term oncofetal RNA for this mode of H19 expression (Ariel et al, 1997a).

The term tumor marker is applied to all substances produced either by the tumor cells or by the host organism in response to a tumor. Its presence may be detected in the serum or other biological fluids or at the level of the tissue, indicating the presence of a neoplasm.

An ideal tumor marker can be used for early detection, diagnosis, differential diagnosis, prognosis, and prediction of response to therapy and follow-up. The possibility of using H19 as a tumor marker was suggested by our group for hepatocellular, bladder, and ovarian carcinomas (Ariel et al, 1995, 1998; Tanos et al, 1999).

More striking is the predictive value of H19 for tumor recurrence (Ariel et al, 2000b). We have found that in transitional cell carcinoma of the bladder with tumors that express H19 in most cells (more than 2/3), median disease-free survival is significantly shorter, only 8.3 months, than in tumors expressing H19 in a smaller number of cells (38 and 51 months for less than 2/3 and less than 1/3 respectively).

Therefore, in the group of patients with refractory superficial bladder cancer, the patients at higher risk of recurrent disease will be those who have more H19-positive cells, and will benefit the most from DNA-based drug based on the transcriptional regulatory elements of H19 (as we will discuss later on). Moreover, it was reported that for all tumors of breast adenocarcinoma displaying a good prognosis (grade I), only the stromal component express H19, while H19 is expressed in both epithelial and stromal components of human invasive breast adenocarcinoma (Dugimont et al, 1995).

One constraint with the use of H19 as a tumor marker relies on its nature of expression, being an untranslated RNA molecule, with no protein product to be detected in the blood or body fluids. However, it can be detected quite easily by in-situ hybridization (ISH) in malignant tissues (Ariel et al, 1997a, 1998). This technique is very sensitive. We were able to detect H19 RNA in a single cell. This could be a useful diagnostic aid because differential expression of H19 is found to have a clinically significant outcome.

B. Setting the stage for a Phase I experiment in patients suffering from superficial bladder cancer using H19 regulatory sequences and DT-A

Cancer is responsible for >20% of all deaths and will soon overtake heart disease as the main killer in the western world. The classical combined treatment of malignant diseases – surgery, radiation therapy and chemotherapy and combinations thereof- often has to face tumors that are already resistant to it. Thus the development of new strategies for the cure of malignant diseases is an evolving field of research, with DNA-based therapeutic approaches being a promising area.

One of the main goals for all cancer therapies is the selective targeting and killing of tumor cells, thus increasing the therapeutic ratio. Both chemotherapy and radiotherapy induce dose- limiting normal tissue toxicities, which reduce their clinical effectiveness.

Cancer based DNA-drug carries the potential to avoid normal tissue toxicity. More importantly, the therapeutic transgene can target tumor cells, an outcome that conventional therapeutic approaches cannot always achieve.

A DNA-based drug for cancer requires the development of delivery systems that allow gene therapy to be administrable to patients in vivo. This requirement demands that the vector itself have some capacity to discriminate between target and non-target cells at some level. If this is not satisfied, dangerous non-specific toxicity could result.

One might avoid this problem by use of vectors that infect or transduce only target cells. However, there are few if any available gene delivery vehicles that allow efficient targeted transduction. Thus, while a relatively high level of infectivity of viral- based vectors remains an attractive feature, there are multiple safety concerns and technical features which limit their application. Consequently, transcriptional targeting is alternative and may remain a required safety feature even in the events of development of reliable transductionally targeted vectors.

Transcriptional targeting depends on specific regulatory sequences (promoters/enhancers) of genes that are identified as expressed or solely up regulated in cancer cells. The investigations of their transcriptional control provide genetic elements whose presence can be targeted in neoplastic cells, resulting in selective death (Abdul-Ghani et al, 2000; Ohana et al, 2002; Ayesh et al, 2003).

Only targeted cells will be able to activate the transcriptional elements due to their processing of unique transcription factors, which drive the expression of the toxin gene. For such an approach to work the promoter elements need to be tumor specific and the regulatory elements of the promoters and enhancers need to be fully characterized. Ideally, tumor specific promoters should be highly active in tumor cells and have little or no activity in normal cells.

The specificity of these strategies should provide improved targeting of metastatic tumors following systemic gene delivery. In the next section we will present a pathway for a phase I clinical trial in bladder cancer using the H19 regulatory sequences and "A" fragment of diphtheria toxin (DT-A). The potential of the toxic vectors driven by the H19 and the IGF2-P3 regulatory sequences have been tested in a metastatic model of rat CC531 colon carcinoma in liver. Preliminary results in treating a patient with hepatocellular carcinoma using H19 regulatory sequences were promising. Preliminary results in treating a patient with colon carcinoma that metastasize to the liver using H19 regulatory sequences and DT-A are promising.

C. H19 and bladder cancer

Bladder cancer is the fourth most common cancer in men, accounting for about 10% of all cancer cases, and is the fifth most common cause of cancer death in men. In woman it is the eighth most common cancer (Silverberg et al, 1990).

Most bladder tumors arise from epithelium lining the urinary system--the transitional epithelium--and are therefore, transitional cell carcinomas. Eighty percent are superficially limited to the bladder mucosa (Ta) or submucosa (T1). These tumors can be removed by transuretheral resection, but tend to recur in 50-70% of all patients.

Attempts were made to decrease the high recurrence rate of superficial bladder tumors by adjuvant intravesical treatments such as intravesical chemotherapy and immunotherapy (BCG). However, a significant portion of patients develop recurrent tumors despite all these efforts, which are also associated with side effects.

Human bladder carcinoma develops in the adult from bladder mucosa cells which have lost their ability to express the H19 gene. In recent years, we have studied the expression of the H19 gene in human bladder carcinoma (Elkin et al, 1995, 1998; Cooper et al, 1996; Ariel et al, 2000a). Human bladder carcinoma is a prototype of tumors, which expresses H19 originating in the transitional epithelium of the urinary tract during fetal life.

We have shown that H19 is significantly expressed in 84% of human bladder carcinomas and that the expression level is linked to the differentiation stage. In a N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) induced bladder carcinoma mouse model, our group has correlated H19 expression, as demonstrated by (ISH), to the sequential stage of tumor progression (Elkin et al, 1998). The mouse model of BBN resulted in the development of invasive carcinoma preceded by pre-neoplastic mucosal changes. This resembles the solid invasive cancer in human.

In this model, the initial expression of H19 was found to be concurrent with epithelial hyperplasia, which appeared as early as 5 weeks following carcinogen ingestion. The localization of H19 expression at this stage is the connective tissue of lamina propria underlying the basement membrane of the hyperplastic epithelium. At 11 weeks some expression was found in the epithelial cells, still in the pre-neoplastic state.

Invasive tumors were first noted at the 20^th week of BBN-treatment. H19 expression was evident at this stage in epithelial tumor cells, in the intervening stroma, and in the submucosa underlying the hyperplastic epithelium adjacent to the tumor. The stroma, the connective tissue that interposes between malignant cells, is an integral part of solid tumors. In contrast, the bladders of the control group that received normal tap water without the administration of BBN, did not express H19 in any of the tissular elements of bladder wall (Elkin et al, 1998).

We have also injected cells from human bladder carcinoma cell lines, which did not express H19, into nude mice. The significant increase in H19 expression during the process of tumor formation in the mice raises the possibility that the H19 gene product fulfills a role in the process of tumor formation of the bladder (Elkin et al, 1995). Moreover, it was reported that LOI of H19 is linked to hypo-methylation of the paternal allele in human bladder cancer (Takai et al, 2001). Differential expression of H19 was found to have a clinically significant outcome in bladder cancers (Ariel et al, 2000 B).

D. Experimental models of bladder cancer "From the bench to the patient"

1. The use of a DNA- based therapy in vitro

Early investigations in any modern anticancer therapy are done in vitro. For this purpose, our team characterized the human and mouse H19 regulatory sequences which can potentially be applied to control the expression of a toxin gene, in constructs to be used in bladder cancer gene therapy trials in mice and humans (Banet et al, 2000). Cloning of different fragments of the human H19 promoter enabled the examination of their transcriptional activity in a variety of cancer cell lines.

The regions between -85bp and -61bp is rich in CpGs, and was found to play a significant role in the regulation of H19 transcription. This region contains transcription factor binding sites, including the CCATT box, which binds transcription factors from the C/EBP family.

We have constructed expression vectors carrying “cytotoxic genes”, such as the gene for the DT-A or the gene of herpes simplex virus thymidine kinase (TK), under the control of an 814 bp 5’ flanking region of the H19 promoter sequence. This region was verified to be highly active in both human and murine cell lines. We also presented evidence that the constructs expressing either the toxin or the suicide gene driven by the H19 regulatory sequence could selectively exert their cytotoxic effects in H19-positive cells (Ohana et al, 2002). The killing capacity of these constructs was in accordance with the relative activity of the H19 regulatory sequences in the transfected cells.

These studies demonstrate that the transcriptional regulatory sequence of the H19 gene can be exploited to achieve high cell-specific expression of exogenous toxin in vitro. The usage of the gene only encoding the diphtheria toxin-A chain prevents any bystander effect because the B chain is responsible for cell penetration. This might be desirable in many cases since it prevents harmful effects to surrounding normal cells.

The potent activity of the toxin can be attenuated by using a mutated DT-A chain gene. Using a plasmid with an expression cassette of the toxin, no immune response will be encountered; moreover, the whole western population is immunized against the toxin. Further, our system will not be affected by a multi-drug resistance effect, a major problem in chemotherapy.

In a second stage of our studies, DT-A toxin expression was evaluated in animal models of bladder cancer. One advantage associated with bladder cancer is its easy accessibility and relative isolation from other areas of the body.

2. Animal models for in vivo DNA-based therapy for bladder cancer

The goal of all laboratory models of cancer is to simulate the human disease. When promising in vitro results are obtained, the next step is animal studies. Most of the animal experimental work in the field of bladder cancer is done on rodents. The road from in vitro experiments to a Phase I clinical study is illustrated, below, using different animal models of bladder cancer.

i. The heterotopic syngeneic mouse model

In this model, malignant cells are injected into the back of the mouse. In an immuno-competent animal a syngeneic cell line is used. The advantages of this model are its rapidity, reproducibility, and visibility of tumors. The disadvantage is the poor correlation in histology and clinical course between this model and the clinical disease. Novel therapies can be tested using this model and it is possible to obtain quick initial results, and use them as a guide for further research.

In this model, syngeneic (MBT-2-t50) bladder carcinoma cells were injected subcutaneously (s.c.) into the dorsa of 6-7-week-old C3H/He female mice. Measurable tumors appeared after 10 days, and treatment began 14 days after inoculation. An H19 gene promoter was used to drive the expression of either the (DT-A) toxin or (TK), followed by ganciclovir treatment, as the therapeutic vector, or the luciferase (luc) gene as a control. Additional controls mice had tumors without any treatment. The results of these experiments showed that the mean weight of the tumors from mice given three treatments of the toxin plasmid was 40% lower than the mean tumor weight of the mice treated with a similar construct expressing the (luc) gene (Ohana et al, 2002).

ii. Heterotopic nude mice model

This model shares the advantages and disadvantages of the syngeneic mouse model. However another advantage of this model is that it can be used to assess the therapeutic potential of our plasmids on tumors formed by cell lines of human origin. We have previously shown that the regulatory sequences of H19 are highly transcriptionally-active in a human bladder carcinoma cell line (RT-112) (Ohana et al, 1999, 2002).

Confluent (RT-112) and (UM-UC3) human bladder carcinoma cells were subcutaneously injected into the back of CD1 nude mice females, 6-8 weeks old. 14 days after cell inoculation the developing tumors were measured in two dimensions and randomized to different treatments.

Two injections of DTA-H19 were able to inhibit any further tumor growth as compared to the control luc-H19 treatment (Ohana et al, 2004a). While the average tumor size of the toxin treated tumors did not change, the control treated tumors continued to increase their volume 2.5 fold during the 7 days after the start of the treatments. The growth rates of treated tumors according to tumor volume measured in vivo were significantly reduced, with relatively large necrotic areas compared to the control.

Moreover, signs of toxicity were not observed. Liver and renal function tests were identical to values obtained from untreated animals. No traces of the plasmid H19-DTA were found in the blood analyzed by PCR after the mice were sacrificed, nor was weight loss of the treated animals observed (Ohana et al, 2004a).

iii. The orthotopic syngeneic models in the mouse and rat

An intravesical tumor is produced by injection of malignant cells (syngeneic cells for immuno-competent animals and human-derived cells for immuno-deficient animals) into the bladder of the rodent. The bladder is rather resistant to implantation of cells, and it is necessary to create abrasions in the bladder mucosa of the anesthetized rodent either by acid, by trypsin installation, or by induction of thermal burns with surgical diathermy, in order to increase "tumor take".

The advantages of this model are its speed, 7-10 days for production of a tumor, and the ability to treat bladder tumors inside the bladder. The disadvantages are the need for anesthesia, and the difficulty in assessing tumor burden. These tumors resemble human bladder tumors but not as much as carcinogen-induced bladder tumors.

Accordingly, a rat model for bladder cancer was developed by intravesical instillation of (NBT-II) rat bladder carcinoma cells onto the wall of rat bladder in vivo. To evaluate the potential use of H19 regulatory sequences for the therapy of this rat bladder carcinoma model, we determined the levels of H19 RNA by PCR, from samples of the developed tumors in vivo 7 days after instillation. It is interesting that the level of H19 RNA was even higher than the original (NBT-II) cells used to establish this model. H19 RNA is undetectable in normal rat bladder tissue, the DTA-H19 construct was shown to be highly active in (NBT-II) cells in vitro. Hence, the validity of using this animal model was shown (Ohana et al, 2004a).

Rats were treated with either the DTA-H19 construct or luc-H19 control four days after (NBT-II) instillation using polycation PEI. Treatment was repeated four days later and rats were sacrificed 11 days after the first treatment. The mean weight of bladders of DTA-H19 treated rats was significantly lower. The average size of DTA-H19 treated tumors was 95% smaller than the luc-treated ones. The group treated with the reporter vector showed more than one large transitional cell carcinoma (TCC) lesion, with different grades of invasion.

In contrast, only smaller papillary tumors were detected in the DTA-H19 treated bladders. No traces of toxicity was seen by blood, urine and weight gain analyses. In vivo evidence of DT-A expression was tested by PCR analysis. There was expression in the tumor, but not in the liver and marginally in the kidney (Ohana et al, 2004a).

iv. Carcinogen-induced bladder cancer in mice and rats

In this model, the rodent is given a carcinogen, most commonly through drinking water. BBN is a carcinogen given in a concentration of 0.05% and it induces bladder tumors in 95% of the rodents after 25 weeks. The tumors produced by the carcinogen resemble human bladder cancer in histology, etiology, and in kinetics (25 rat weeks equal 10 human years- the expected incubation period of human bladder cancer).

Treatment with DTA-H19 constructs resulted in about 50% reduction of tumor area and large necrotic areas as detected by histological examination, while no significant reduction of the weight of the bladders was noted between DTA-H19 and Luc- H19 constructs. (Unpublished results).

v. Treatment of two human patients with the therapeutic vector DTA-H19

These patients suffered from refractory superficial bladder cancer. They developed recurrent bladder tumors and failed to respond to all acceptable treatments known to inhibit the development of recurrent tumors. They were also candidates for cystectomy.

Samples of bladder tumors from both patients were removed by transuretheral surgery. The samples were submitted for histological and molecular diagnosis. In-situ hybridization studies showed high expression of H19 in their bladder tumors. Hence, they were appropriate candidates for the treatment with the toxin construct DTA-H19 (Ohana et al, 2004a).

Taking advantage of the anatomy of the bladder, we evaluated the feasibility and safety of intravesical vector instillation using a transurethral catheter. The experiments on human patients were promising and supplied plenty of important information. The most prominent points are:

1- Intravesical instillation of DTA-H19 construct in complex with PEI is safe up to a dose of 5mg in a single treatment. No adverse side effects related to the constructs occurred during 30 treatment sessions in the two patients (20 treatments to the first patient, and 10 to the second).

2- The DTA-H19 construct is not absorbed into the blood stream. This was supported by the results showing no trace of DTA-H19 plasmid in the blood even after extensive PCR cycling analysis done on blood samples regularly taken during the course of treatment before and 2 hours after plasmid instillation of plasmid administration.

3- Evidence for biological activity of the transgene was strongly demonstrated by video-cystoscopy. The treatment with the DTA-H19 construct has an ablative power on bladder tumors with > 75% decrease in tumor volume, as compared to the video-cystoscopy before treatment, and was coupled with macroscopic signs of tumor necrosis.

4- The DTA-H19 plasmid is excreted in the urine up to one week after instillation as determined by PCR analysis. Although clearance of the plasmid was observed with time, the presence of the plasmid in the urine for a prolonged period is a great advantage, because the plasmid may be continuously available to transfect deep layers of tumor once the superficial layers of the tumor have been transfected and pilled off.

5- The source of the DTA-H19 plasmid excreted in the urine after the instillation comes both from exfoliated cells and from the supernatant (probably plasmid loosely adherent to outer cell surface). The plasmid DTA-H19 probably enters the nucleus since the plasmid presents in the extract of genomic DNA from the cells pelleted from the urine.

6- In the first patient, no recurrence of TCC occurred even 17 months after the first DTA-H19 plasmid treatment, while no recurrence occurred even after 14 months in the second patient.

In conclusion, these points show that bladder tumors may be successfully treated by a DNA-based drug approach based on transcriptional targeting using the H19 gene regulatory sequence. This treatment seems safe, very efficient, promising, and above all can increase the quality of life of many patients. Our study may be a platform for the design of extensive phase I and II studies on a larger number of human patients, and may be a major turning point in the treatment of human bladder carcinoma (Ohana et al, 2004a).

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

We extended the described approach to colorectal rat liver metastases. The therapeutic potential of the toxin vectors driven by the human H19 and IGF2-P3 regulatory sequences was tested in a metastatic model of rat (CC531) colon carcinoma in liver (Ohana et al, 2004b).

We have already characterized the expression profile of H19 in human liver metastasis originating from colorectal, pancreatic, ovarian, breast and gastric cancers. H19 is highly expressed in various human liver metastases as detected using quantitative (ISH) analysis (accepted results for publication). Moreover, we have previously described the construction of expression vectors carrying the diphtheria-toxin A-chain gene under the control of IGF2-P3 and P4 promoters, and showed that these constructs selectively kill tumor cells and inhibit tumor growth in vitro and in vivo (Ayesh et al, 2003).

In order to determine the validity of this approach for therapy of rat colon tumors in the liver, the level of H19 transcript in normal rat and tumor livers induced by subcapsular injection of CC531 cells was detected , results showed high levels of H19 RNA in liver metastasis, in contrast, H19 RNA levels in normal liver or liver parenchyma that was not involved in metastasis were marginal. Moreover, high levels of P2 transcripts were also detected in the metastatic tissue where no or very low expression levels were detected in the adjacent host liver tissues by using (ISH) technique (Ohana et al, 2004b).

The pattern of rat igf2-P2 expression in the rat liver resembles that of the human IGF2-P3 expression (Kawamoto et al, 1999). We previously reported that the human H19 and IGF2-P3 regulatory sequences are also active in murine cells (Ohana et al, 2001; Ayesh et al, 2003). We also showed that human H19 and IGF2-P3 regulatory sequences were able to drive the DTA expression in orthotopic rat colon carcinoma (CC531) cells.

Intratumoral injection of the toxin vectors induced a 90% and 50% reduction in the medium tumor volume for H19 and IGF2-P3 therapeutic vectors respectively, as compared with the control groups that were treated with the correspondent reporter vectors. The therapeutic effect was accompanied by an increase of necrosis of the tumor. No signs of toxicity were detected in healthy animals after their treatment by the toxin expressing vectors. Although liver metastases were not totally ablated, the treatment with the toxin vectors almost stopped the further growth of the metastasis (Ohana et al, 2004b).

The inhibition in tumor growth was also demonstrated in a group of rats treated with H19-DTA, compared with a control group, in a long-term experiment. The decrease in medium tumor volume in this experiment, 43%, was smaller than that obtained in a short-term experiment. The reduced efficiency of the long term experiment may be due to the longer time intervals between treatments with the plasmids (7 days compared with 4 days, to avoid complications due to surgical procedures).

Based on the findings presented here, we propose a patient-oriented approach to treat colon metastases in the liver. Given the clinical and genetic diversity of liver metastases, it may prove difficult to find a one-for-all promoter sequence to be used as a therapy.

Instead we propose tailored transcriptional regulatory sequence selection for DNA-based therapy of liver metastases, according to an individual patient-specific gene expression profile. According to this approach, the patient may be simultaneously treated with a combination of toxin-expressing plasmids, some of which are under the control of the H19 and IGF2 regulatory sequences, and others depending on differentially expressed gene profiling.

We recently used regulatory sequences of H19 to drive the expression of DT-A in treating a patient with colon carcinoma that metastasizes to the liver. Preliminary results are promising.

V. Proposed mechanism of action of H19 RNA

The goal of uncovering the H19 gene function has led to many investigations. Early attempts worked with models of H19 over-expression and knockout in mice. When extra copies of the H19 gene were introduced into mouse embryos, no transgenic progeny were obtained which expressed the transgene, and fetal death occurred at midgestation. However, in the developing embryo before death, the transgene was expressed in several tissues including those in which the endogenous gene is normally not expressed (brain tissue) (Brunkow and Tilghman, 1991).

When H19 was knocked out creating a null mutation for the H19 gene, leaving the 5' enhancer region intact, the paternal transmission of the mutation neither exhibited any obvious phenotype nor aberrant H19 expression (Leighton et al, 1995). This was consistent with the inactivation of the paternal allele under physiological conditions. However, the maternal transmission of the mutation showed a 28% increase in the body size that could be attributed to the higher levels of IGF2.

Despite much evidence, uncovering the H19 gene function remains elusive. We have made contributions that could lay the foundation for understanding more about this gene. H19 is a stress regulated oncofetal RNA. H19 seems not to contribute to the onset stages of tumor development, but can be added to the list of genes that dictate a tumor's deadly signature (Ayesh et al, 2002, 2004).

A. Functional grouping of genes modulated by H19 RNA: trends to cellular migration, angiogenesis and metastasis

An important step towards understanding the role of H19 RNA is defining gene expression profile; comparing patterns of gene expression in two homogeneous cell populations that only differ in the presence or absence of H19 RNA. This approach for analyzing the expression of multiple genes simultaneously using the cDNA expression array, has widely opened the way to unmasking the function of this gene.

Plenty of downstream effectors of H19 RNA have been identified and among these are group of genes that were previously reported to play crucial roles in some aspects of the tumorigenic process. H19 RNA presence could enhance the invasive, migratory and angiogenic capacity of the cell by up regulating genes that function in those ways, and could thus contribute at least to the initial steps of metastatic cascade (Jiang et al, 1995; Abramovitch et al, 1998; Arkonac et al, 1998; Kacimi et al, 1998; Giroux et al, 1999; Sebolt-leopold et al, 1999; Huang et al, 2000; Schlessinger et al, 2000; Wang, 2001) (Figure 2).

Previous observations made by us and others are in accordance with this proposal. First, it was demonstrated that halofuginone, a low molecular weight alkaloid compound known to inhibit collagen type a-1 and MMP-2 gene expression, effectively suppresses the progression of primary tumors in both transplantable and chemically induced models of bladder cancer. It abrogated BBN-induced tumor in the mouse bladder, and its progression towards highly invasive carcinoma. Moreover, a significant reduction in vascular density was revealed by histological examination of tumors derived from halofuginone-treated mice (Elkin et al, 1999). All these observations are coupled with the absence of H19 gene expression in halofuginone-treated mice as compared with its high level in BBN-induced bladder tumors in mice that did not received halofuginone.

ISH revealed a high level of H19 expression in both epithelial and stromal compartments of bladder tumors induced by BBN. After halofuginone treatments, H19 expression was not detected in any tissue element of the bladder wall (Elkin et al, 1999). Taking into consideration that the course of H19 expression in BBN-induced bladder cancer in mice is essentially the same as in humans (Elkin et al, 1998), lack of H19 transcripts in halofuginone-treated bladders could be related to the observed anti-invasive and anti-angiogenic effect of halofuginone. However, the direct effect of halofuginone on H19 expression needs to be elucidated.

Second, it is well established that epithelial-mesenchymal transitions are vital for morphogenesis during embryonic development and are also implicated in the conversion of early stage tumors into invasive malignancies. (Adriaenssens et al, 2002) showed that cross-talk between mesenchyme and epithelium increases H19 gene expression during scattering and morphogenesis of epithelial cells. The mesenchymal factor hepatocyte growth factor also known as scatter factor (HGF/SF), was capable of inducing H19 expression and cell morphogenesis.

This finding places H19 as a target gene for HGF/SF, and suggests that up regulation of H19 may be implicated in morphogenesis and/or migration of epithelial cells (Adriaenssens et al, 2002). Moreover, it was shown that the H19 promoter region is responsive to HGF/SF in a transient transfection assay. However using pharmacological inhibition of ERK/MAPK prevented the activation of H19 promoter in response to HGF/SF. This study highlights H19’s potential role in promoting cancer progression and tumor metastasis by being a responsive gene to HGF/SF.

The study also showed that H19 is often over-expressed in stromal cells and preferentially located at the epithelium/stroma boundary; in some cases of breast adenocarcinoma with poor prognosis, H19 is over-expressed in epithelial cells. Moreover, regulation and general expression patterns of H19 in the mammary gland during fetal or postnatal life may argue in favor of the H19 being involved in epithelial cell migration. Indeed in puberty and pregnancy, H19 is strongly expressed in mammary terminal buds extending into the fat pad and the mesenchymal components (Adriaenssens et al, 1999).

It was shown that the well-differentiated breast carcinoma cell line MCF-7 does not express the c-Met protein (the receptor of HGF/SF). Therefore it does not demonstrate a motile or invasive phenotype; nor is H19 induced by HGF/SF in this cell line. In contrast, poorly differentiated cell lines (MDA-MB-23, and HBL-100), that are sensitive to HGF/SF, exhibit a high level of H19 gene expression. This is concordant with a fully developed invasive phenotype (Adriaenssens et al, 2002). In a related way, it was very recently reported that the level of H19 expression is strongly correlated with tumor invasion and myometrium invasion of the female genital organs (Lottin et al, 2005).

Relevant to the physiological role played by H19 RNA, many features of H19 expression during embryogenesis and adult life are in accordance with our proposed function of H19 RNA. It is very interesting to note that while H19 expression is down regulated after birth, the endometrium and the ovary of the human female are two of the very few tissues which retain the ability to express the H19 gene in adult life (Ariel et al, 1997b). H19 has been shown to be present in the secretory endometrium of the female menstrual cycle, which is characterized by increased vasculature. It is also known that physiological angiogenesis rarely occurs in adults, but does occur during menstruation for repair of the vascular bed.

Furthermore, H19 was found to be expressed at high levels during embryogenesis, and the above-proposed activity of H19 can explain the role of H19 in placental and embryonic development. H19 is reported to be highly expressed in extra-villous trophoblasts cells, which are responsible for the invasive properties of the placenta during implantation (Ariel et al, 1994). Moreover, H19 expression increases in the carotid artery after injury suggesting a role during wound healing (Kim et al, 1994), where angiogenesis is fundamental to restoring or creating a blood supply to growing tissue.

B. H19 is a stress regulated gene, with growth benefit in serum stress, and is up regulated in response to hypoxia and/or serum starvation

The involvement of H19 in cell cycle control was previously predicted by the involvement of the tumor suppressor gene p53 in the down regulation and promoter activity of H19 expression, which lacks a p53 consensus site and a TATA box (Dugimont et al, 1998). We previously provided experimental evidence for a role played by H19 in enabling the cells to overcome the stress caused by serum starvation. We indicated that while H19 does not confer any growth advantage on cells cultured in 10% fetal calf serum (FCS), it does enable them to continue proliferating when the cells are cultured in a serum stressed media (0.1%FCS) for 72 hours (Ayesh et al, 2002).

This observation was coupled with the inability of the H19 expressing cells to induce the cdk inhibitor p57Kip2 in response to serum stress. This observation conditionally (absence of serum factors) places H19 gene in the core mechanism of cell cycle control and cell proliferation, especially at the transition from G1 to S phase and thus could indicate the importance of H19 gene expression to the tumor under stress conditions, like when the growth factors availability is strict (Ayesh et al, 2002).

Taking a look at the set of genes modulated by the presence of H19 RNA, we found that uPAR and ERK1/2 is up regulated. The growth advantage of H19 expressing cells under serum starvation could be related to the up regulation of those genes.

Recently we showed that H19 is a responsive gene to hypoxia with or without serum starvation, depending on the cell line under investigation. Plenty of downstream effectors of H19 under hypoxic conditions and/or serum starvation have been identified, which further indicates a role of the H19 gene in the initial steps of metastatic cascade (Ayesh et al, 2004). All of these observations can place the H19 RNA into the category of biotic and abiotic stress response RNA.

We thus hypothesize the possible role played by H19 RNA in tumorigenicity. During tumor development, an evolution-like process occurs in which cells with highly advantageous properties are selected. In this regard, the microenvironment of the tumor is the factor that imposes the selective pressure on the malignant cells. The ability of the cells to withstand harsh conditions due to the lack of adequate blood supply is of great advantage for tumor progression. Populations of cells may be forced into a state of dormancy, rendering themselves non-proliferative, and hence lowering down their needs for oxygen and metabolites.

In contrast, any genomic alternation, in our case increased in H19 expression, occurring in a malignant cell which enhances its ability to proliferate under harsh condition, will give that cell a selective advantage. The progeny of the H19 expressing cells will increase in number at a higher rate than the neighboring cells and hence gradually increase their relative contribution to the tumor volume.

Phenotypic traits beneficial for cell proliferation also seem to favor invasive growth and metastasis. Less sensitive cells in a harsh and stressed condition will indeed contribute greatly to the generation of a patho-physiological microenvironment with harsher low extracellular pH (more acidic) and low oxygen tensions (hypoxia). Experimental studies have shown that hypoxic tumors may also be the most pro-angiogenic, and pro-metastatic. Hypoxic conditions in tumors induce the release of cytokins that promote vascularization and thereby enhance tumor growth and metastasis (Brahimi-horn et al, 2001; Harris, 2002; Choi et al, 2003).

Since we provide evidence that H19 RNA enables the cells to continue proliferating in a serum starved condition (one of the consequences of a poorly vascularized tumor), it is logic to assume that the relative contribution of H19 expressing cells towards the creation of the pathological microenvironment is great, which will promote for tumor angiogenesis, invasion and metastasis. Here, it seems that H19 have dual effects on the accelerated creation of the pathological environment, and the modulation of some of the genes that are required for hypoxic response needed for angiogenesis and metastasis (Figure 3).

This proposal is supported by many observations:

1. It was previously shown by us that while H19 RNA is either not or weakly expressed in certain bladder carcinoma cell lines in normal culture conditions, H19 is strongly expressed when tumors are grown by injecting these cell lines into nude mice (Elkin et al, 1995). Although we can’t rule out any other possibility, it seems that enhanced H19 expression observed in these tumors has to do with selection and clonal expansion of H19 expressing cells, under the severe and harsh condition of a rapidly growing tumor in vivo. This explanation is strengthened by our observation that these cells have lost H19 expression upon the third passage when re-cultured in vitro and provided with essential nutrients and serum factors and oxygen.

2. The patients with the most hypoxic tumors had worse disease-free and overall survival probabilities than those with the least hypoxic tumors, irrespective of whether the treatment was a surgery or radiation therapy. Moreover, recurrent tumors were found to show

significantly lower median pO2 values than primary tumors of comparable size, as has been reported for uterine cervix (Hockel et al, 1993).

Consistent with this, our group showed that H19 gene expression is a marker of early recurrence in human bladder carcinoma. Moreover, we have found that in the group of patients with refractory superficial bladder cancer, those that have more H19 positive cells are at higher risk of recurrent disease, and significantly shorter median disease-free survival (Ariel et al, 2000b).

A proteomic approach has revealed that H19 over expression in human cancerous mammary epithelial cells stably transfected with genomic DNA containing the entire H19 gene, is responsible for positively regulating the thioredoxin gene at post-tramscriptional level, thioredoxin being a key protein of the oxidative stress response and deoxynucleotide biosynthesis (Lottin et al, 2002b).

3. Many processes that involve cellular invasion, including blastocyst implantation, and placental development occur in reduced oxygen environments (Rodesch et al, 1992). These two physiological processes show intensive up regulation of H19 expression (Ariel et al, 1994).

4. Comparison of the tumorigenic potential of the human epidermoid carcinoma cell (HEp3) which expresses uPAR, and its variant in which uPAR expression was reduced by an antisence strategy, reveals that reduction of uPAR expression forces malignant cells into a protracted state of dormancy. The uPAR-expressing clones produced rapidly growing, highly metastatic tumors within two weeks of inoculation on the chorioallantoic membranes (CAMs) of chick embryo. In contrast, each of the clones with low surface uPAR, whose proliferation rate in culture was indistinguishable from controls, remained dormant for up to 5 months when inoculated on CAMs. Calculating the percent of apoptotic cells and S-phase cells in vivo showed that the mechanism responsible for the dormancy was diminished proliferation (Yu et al, 1997).

It was speculated that reduced uPAR might diminish responses mediated via this receptor, such as migration, mitogenicity, and induction of neovascularization leading to tumor dormancy. As we stated above, uPAR expression is up regulated by H19 RNA.

5. It is very interesting to note that several genes up regulated in the presence of H19 RNA are also known to be induced by hypoxia. For example, it was reported that hypoxia increased in vitro invasiveness of trophoblasts and breast carcinoma cells through the increase in uPAR expression, and decreased adhesion of the cells to fibronectin through reduced expression of a-5 Integrin (Lash et al, 2001). Those observations are similar to what we observe in the presence of H19, where uPAR expression is induced and Integrin a-5 is reduced. Moreover, the transcription factor NF-kB, as well as members of the mitogen-activated protein kinase (MAPK) superfamily, consisting of (ERK)1/2, c-Jun, NH2-terminal kinase (JNK1/2), all induced by the presence of H19, have been reported to be activated by hypoxia (Laderoute et al, 1999; Kroon et al, 2001). TNF-a, IL-6, ICAM-1 (Kacimi et al, 1998), c-Src (O'toole et al, 1997), Ezrin (Mukhopadhyay et al, 1995), HB-EGF (Sakai et al, 2001) and Transferrin receptor (Tacchini et al, 1999) are also induced by hypoxia and are up regulated by H19. We recently provided evidence that H19 itself is a hypoxia responsive gene (Ayesh et al, 2004).

6. H19 RNA has been detected in rheumatoid arthritis synovial tissue (Stuhlmuller et al, 2003). Although this provides one of the first cases of H19 expression in chronic, non-tumoral disease, the hyperplastic synovial tissue in rheumatoid arthritis displays several features of a semi-transformed tissue, such as invasive growth into cartilage and bone. Moreover, the presence of extensive angiogenesis is usually associated with rheumatoid arthritis due to hypoxic and oxidative stress, partly due to the metabolic activity of increased inflammatory cell exudates in the affected area. This could explain why H19 was detected in rheumatoid arthritis synovial tissue.

What is common to serum starvation and hypoxia? Hypoxia and serum starvation are both the consequence of a poorly vascularized tumor, which is considered a normal stage during tumor development. The realization that a lot of us carry in situ tumors, but do not develop the disease, suggests that these microscopic tumors are mostly dormant and need additional signal to grow and become lethal tumor (Folkman and Kalluri, 2004). In the absence of blood supply in situ tumor can remains dormant indefinitely. Our findings could mean that H19 expression not only confers a growth advantage on cells under a stress condition (serum starvation), but also up regulates the expression of genes that could contribute to the promotion of cancer progression and metastasis, and thus could be one of those signals that dictate tumors deadly signature.

In conclusion, the oncofetal H19 RNA is a unique gene that can be used for cancer diagnosis as well as therapy. The 33 types of human cancers that express H19, the simplicity of the H19 construct therapy, the promising outcomes of our animal experiments using this therapy, and the promising outcomes and absence of side effects in our limited human trial using this therapy, all point to the value of a therapognostic strategy as part of the arsenal to win the war against cancer.

Acknowledgements

We thank Avi Barak president and C.E.O of Yissum technology transfer company of the Hebrew university of Jerusalem for his kind support and to Dr. Rachel Hochberg for her critical reviewing and to the Israeli ministry of science through its National Knowledge Center in Gene Therapy at the Hadassah-Hebrew University in Jerusalem as well as by grants from the Grinspoon, Blum and the Horwitz Foundations for financial support.

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Imad Matouk Abraham Hochberg

Review Article

Received: 24 February 2005; Accepted: 8 March 2005; electronically published: April 2005

I. Introduction

Acknowledgements

References