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1.  Introduction

1.1. Multi-Step Progression of Tumors

Experimental approaches, cytogenetic observations and molecular analysis have shown that tumors result from a subversion of diverse mechanisms controlling growth, division, and mortality of cells. Tumor development is generally considered as a multi-step process including consequent dysfunction of genes classified into three categories (Bishop, 1995):

  1. proto-oncogenes, which are activated by mutations and become oncogenes. Their acquired oncogenic functions lead to uncontrolled cellular growth and proliferation.
  2. tumor-suppressor genes, which normally negatively regulate cell growth and division preventing the development of tumor. Loss or mutational inactivation of these genes leads to the deregulation of cell cycle progression, and other intracellular processes resulting in cancer progression.
  3. genes involved in maintaining the genomic stability and genes encoding the DNA-repair system. The “loss-of-function” mutations of these genes result in genetic instability characteristic for tumor cells.

1.1.1. Oncogenes

The oncogenes are genes that are capable of stimulating cellular growth. Their precursors (proto-oncogenes) are present in eukaryotic cells, and promote the normal growth and division of cells. Their oncogenic potential can be activated by one of the following mechanisms (Bishop, 1991). 1 - point mutation or chromosomal rearrangement resulting in an abnormal protein which has a different biological activity, for example, RasV12 (Satoh et al., 1992), and Bcr-Abl (Wang, 1988). 2 - gene amplification increasing the number of copies of a normal proto-oncogene within a cell leading to the activation of its oncogenic potential, like MYCN (Schwab, 1990). 3 - viral infection resulting in the control of a proto-oncogene by a more active viral promoter (Lipsick and Wang, 1999).

1.1.2. Tumor Suppressor Genes

The products of tumor suppressor genes normally negatively regulate cell growth (Schwab, 2001). Loss of one or several tumor suppressors is required for the full tumorigenic conversion of a normal cell. Re-expression of these genes in malignant cells leads to the restoration of growth regulation and the reversion of the transforming phenotype (Klein, 1998).

First category of these genes consists of known tumor suppressors down-regulated in transformed cells through chromosomal deletion, loss of heterozygosity (LOH), and mutagenesis. Positional cloning is a classical approach of molecular genetic to identify this kind of genes.


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To analyse a difference between normal and tumor cells in more detail, new methods such as differential gene cloning (Lau and Nathans, 1985) or subtractive hybridisation (Scott et al., 1983), and improved second generation like differential display (Lang and Pardee, 1992) subtractive suppression hybridisation, SSH (Diatchenko et al., 1996) were developed. Analysis and comparison of the expression patterns of normal and tumorigenic cells using these methods revealed a new category of genes down-regulated but not mutated in tumor cells. This observation led to the postulation of two classes of the tumor suppressor genes (Sager, 1997).

class I tumor suppressor genes which inactivated by chromosomal rearrangements like a deletion or translocation, and by a mutation of one or both alleles (Hanahan and Weinberg, 2000).

class II tumor suppressor genes which are stably down-regulated but were not found to be mutated in significant subset of cancers and cancer cell lines (Sager, 1997).

1.1.2.1. The Class I Tumor Suppressor Genes

Since the first tumour suppressor was identified, more than 20 other genes have been shown to be mutated or deleted in tumours, inter alia pRB, p53, WT1, BRCA1, BRCA2, APC, NF1, and NF2 (McCormick, 2001; Schwab, 2001). The genes disrupted in a majority of human cancers are the retinoblastoma tumour suppressor gene (RB1), and the TP53 gene. The breast cancer susceptibility genes 1 and 2 (BRCA1 and BRCA2) were demonstrated to play an important role in the heredity of ovarian and breast cancers (Beckmann et al., 1997).

The RB1 gene was the first tumor suppressor gene to be isolated and cloned (Friend et al., 1988). The product of this gene, the retinoblastoma protein, pRB, is a nuclear phosphoprotein which mediates progression through the first phase of the cell cycle, playing a major role in the control of cell division and differentiation (Cordon-Cardo et al., 1994). Cytogenetic studies of chromosomal alterations in a childhood retinoblastoma, and in breast, lung and pancreatic cancers demonstrated a correlation between tumorigenesis and chromosomal aberrations on chromosome 13q14 where the RB1 gene is located (Michalova et al., 1982). The inactivation of one of the RB1 alleles by point mutation or deletion was demonstrated to be often accompanied by loss of heterozygosity (LOH) on chromosome 13 (Lee et al., 1988; Hesketh, 1997). In order to explain the nature of retinoblastoma formation, Knudson suggested the so called “two-hit hypothesis” (Knudson, 1971). He proposed that two inactivating mutations affecting both copies of a gene are necessary for retinoblastoma development. The first could be either a germline or somatic mutation, whereas the second mutation is always somatic.


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This hypothesis illustrated how somatic and inherited mutations might collaborate in tumorigenesis, and also proposed that mutations of tumor suppressor genes have a recessive character, behave recursively at the cellular level.

The TP53 tumor suppressor gene was demonstrated to carry homozygotic somatic alterations in roughly 50% of all human tumours. Mutations in the single copy TP53 gene are the most frequent genetic changes yet shown in human cancers and occur in 70% of all tumors. Germline mutations of the gene have been shown to be associated with the Li-Fraumeni syndrome (Levine, 1997). Further investigations revealed that in contrast to the pRB protein and Knudsons “two-hit hypothesis” in some tumours the TP53 gene carries mutations leading to cancer in a dominant negative fashion (Brachmann et al., 1996).

The TP53 gene encodes a transcription factor activated in response to physical or chemical stress. The p53 protein controls induction of apoptosis, cell cycle progression into G1 and G2 phases, modulation of DNA replication and repair, preventing proliferation of cells with damaged genetic material. Overexpression of wild type TP53 in different cell types leads to growth inhibition (Casey et al., 1991), or to the induction of apoptosis in squamous carcinoma cell lines (Liu et al., 1995). The major down-stream p53 effector participating in the control of the cell cycle check-points is the cyclin dependent kinases inhibitor p21WAF1, functioning as a tumour suppressor itself (el-Deiry et al., 1993; Sheikh et al., 1994). Other p53 effectors playing a critical role in apoptosis signalling are the death signalling receptor Apo-1/Fas (el-Deiry, 1998), the repressor of apoptosis Bcl-2, its inhibitor BAX-1 (Sheikh et al, 1994), and the death receptor DR5 (Burn et al., 2001). Several members of the DNA repair machinery, for example, auxiliary subunit of polymerase δ (PCNA), and replication protein A (RPA) were also described as p53 targets (Schwab, 2001).

1.1.2.2. The Class II Tumor Suppressor Genes

The class II of tumor suppressors is represented by genes which, unlike class I, are not mutated during tumorigenesis but rather have sustained a blockage of their expression through diverse mechanisms (Sager, 1997). Interestingly, that some genes exhibit features of class II tumor suppressors in one type of cancer, whereas in other type they are known to belong to the class I tumor suppressors. Thus, allelic loss of IRF1 occurs frequently in the acute myeloid leukemia, myelodysplastic syndrome (Boultwood et al., 1993), and gastric cancer (Tamura et al., 1996), whereas in ovarian cancer IRF1 is described as a class II tumor suppressor (Sers et al., 2002). Dysfunction of maspin via mutation was identified in prostate cancer (Umekita et al., 1997), whereby down-regulation of this gene is characteristic for many other tumors where the gene is not mutated (Sager, 1990).


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An important feature of the class II tumor suppressor genes is that their down-regulation is reversible. The normal genes are present, and their re-expression might be induced by drugs or other treatments, that makes such genes attractive targets for cancer therapy. It is known that inactivation of the expression takes place on the transcriptional and translational levels during cancer progression. But until now the mechanisms of the gene silencing have not yet been elucidated in much detail.

1.1.3. Mechanisms of Gene Silencing

1.1.3.1. DNA Methylation and Deacetylation

One of the important mechanisms of gene silencing is DNA methylation. The nonmethylated CpG islands within promoter regions were demonstrated to be primary targets for the aberrant hypermethylation in tumour cells (Bird, 1995). Loss of expression, associated with the hypermethylation of the promoter CpG islands, was shown for the RB1 gene in 10% of the patients with the sporadic form of retinoblastoma (Greger et al., 1994). Methylation of WT1 and calcitonin appears in 68-74% of the analysed colon carcinomas (Hiltunen et al., 1997). The p21 WAF1, APC, p15/INK4B, and p16/INK4 genes were also described to be methylated in tumours (Cameron et al., 1999; Baldwin et al 2000; Roder et al., 2002). Interestingly, for several tumor suppressor genes like BRCA1, RB1, and p16/INK4, methylation was described as an additional mechanism of down-regulation in these types of cancer, where the genes are only rarely mutated (Garinis et al., 2002).

The de novo methylation has two consequences: first, it leads to the inhibition of transcription factors binding (Baylin et al., 1998). Second, methylation attracts other proteins that specifically bind the modified DNA. This blocks an access of the transcription factors required for gene expression to DNA (Bird, 1995), and induces a secondary DNA modification, histone deacetylation, resulting in DNA compaction. This process makes DNA less accessible for the transcriptional machinery (Rountree et al., 2000).

Reactivation of tumor suppressor gene expression often required both de-methylation, and inhibition of histone deacetylation, suggesting that these two processes act synergistically in gene silencing, and may both contribute to oncogenesis and cancer progression (Cameron et al., 1999).

1.1.3.2. Inhibition of Positive Regulators of Transcription

Inactivation of the class I tumor suppressors by mutations or deletions leads to a reduced expression of downstream target genes. The first evidences for such an indirect inactivation of tumour suppressors was described for the p21 WAF1 gene down-regulated after loss of p53 due to mutations in the TP53 gene in a variety of human malignancies and cancer cells lines (Shiohara et al., 1994).


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Further investigations demonstrated that the loss of p53 results in the down-regulation of many other tumor suppressors, such as the signaling regulator caveolin-1 (Bist et al., 2000), the angiogenesis inhibitor thrombospondin (Dameron et al., 1994), and serine protease inhibitor maspin (Zou et al., 2000). The tumor suppressor gene maspin was originally identified in normal breast epithelialcells (Zou et al., 1994). Further investigation demonstrated down-regulation of maspin in breast, prostate and colon tumors (Zou et al., 2000; Umekita et al., 1997). Investigation of maspin expression revealed a correlation between its down-regulation and p53 inactivation that led to a proposition that maspin might be a target of p53. The hypothesis was confirmed by the fact that over-expression of wild type p53 in prostate and breast cancer cells led to the rapid induction of the maspin expression via direct binding to the promoter sequence (Zou et al., 2000). Thus, tumor development includes the inactivation of class I tumor suppressors such as p53, followed by the down-regulation of their down-stream target genes, the class II tumor suppressors.

1.1.3.3. Inhibition of Expression by Activation of Oncogenic Signaling

Activated oncogenes might also suppress the transcription of negative growth regulators. Thus, the oncogene Myc, activated in a large number of human cancers (Adams and Cory, 1992), was demonstrated to act not only as a transcriptional trans-activator but also to mediate down-regulation of a variety of genes (Kato et al., 1990). The mechanism of a direct transcriptional repression by Myc is poorly understood, but resent data suggest that Myc mediates repression via negative interference with transcriptional coactivators. Thus, expression of the tumor suppressor p15INK4b is repressed by Myc through association with Miz-1 transcriptional factor (Staller et al., 2001). The alternative mechanism of Myc-mediated suppression of transcription is an association with transcriptional repressors and recruitment of histone deacetylases to the promoter region of target genes (Satou et al., 2001).

Oncogene Ras negatively regulates expression of a number of genes not via a direct inhibition of transcription but through the activation of its down-stream effectors, such as AP1 transcriptional factor. Activation of AP1 results in the negative regulation of various tumor suppressors, for example p21WAF1 (Chang et al., 2002).

To identify genes, expression of which is suppressed via activation of the RAS-downstream signaling, a comparison of the expression patterns of the non-tumorigenic rat fibroblasts 208F and its malignant HRAS-transformed derivative, FE-8, was performed (Zuber et al., 2000). Stable down-regulation of a significant subset of class II tumor suppressors was identified. Expression of a fraction of these genes was recovered in the FE-8 cells after inhibition of the MEK1 kinase, a downstream effector of HRAS, via addition of PD 98059. Re-expression of cdc21 (Mcmd4), lysyl oxydase (Lox), STAT5a, and other genes was obtained (Zuber et al., 2000).


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Down-regulation of lysyl oxidase in many human tumors has been reported, but mutation of the lysyl oxidase encoded gene (Lox) was described only in colorectal tumors, suggesting that lysyl oxidase belongs to the class II tumor suppressors (Csiszar et al., 2002).

Interestingly, inhibition of the lysyl oxidase expression in rat kidney fibroblasts led to the development of the transformed phenotype, activation of the Ras oncogene, anchorage independent growth, and tumorigenicity in nude mice (Giampuzzi et al., 2001). Resent investigations demonstrated that overexpression of lysyl oxidase inhibits Ras-mediated transformation by prevention of NF-kappa B activation, highlighting its particular role in controlling Ras activity (Jeay et al., 2003), and confirming its role as a class II tumor suppressor in the reversion of the malignant phenotype.

Rat H-rev107 was identified as a gene down-regulated in HRAS transformed fibroblasts, and up-regulated in revertant and transformation-resistant fibroblasts (Hajnal et al., 1994). Further investigation demonstrated that H-rev107 possesses growth and tumor-inhibitory capacity and, therefore, belongs to the class II tumor suppressors (Sers et al., 1997). Interestingly, similar to the lysyl oxidase gene, down-regulation of H-rev107 was found to be reversible. Recovery of its expression was obtained in KRAS transformed rat ovarian surface epithelial cells after inhibition of MAP/ERK signalling pathway (Sers et al., 2002). The human H-REV107-1 gene and its related gene H-REV107-2/TIG3/RIG1 were cloned several years ago (Husmann et al., 1998; Di Sepio et al., 1998). Both were demonstrated to possess transformation suppressive properties. H-REV107-1 was shown to be implicated in IFNγ signaling, and its expression was recovered in ovarian carcinoma cells after induction with IFNγ (Sers et al., 2002). H-REV107-2/TIG3/RIG1, originally isolated from retinoid-treated cultured epidermal keratinocytes, was demonstrated to participate in the retinoic acid signaling, and negatively-regulate c-Jun N-terminal kinase and p38 mitogen-activated kinase (Huang et al., 2002). In the meantime it is known that an H-REV107-like subfamily of proteins is exists, and consists of 5 members, which were demonstrated to be down-regulated in various human tumors. Their functions have not yet been elucidated in much details, but resent phylogenetic analysis of the NlpC/P60 protein hydrolases demonstrated that H-REV107-like proteins belong to this superfamily (Anantharaman and Aravid, 2003).

1.2. H-REV107-1 is a Member of the NlpC/P60 Protein Superfamily

1.2.1. The NlpC/P60 Protein Superfamily

The H-REV107-like proteins harbour the NlpC/P60 domain specific for bacterial peptidases. Phylogenetic analysis revealed a large superfamily of proteins related to the E. coli lipoprotein NlpC, and possessing the so called NlpC/P60 catalytic conservative domain, essential for the hydrolytic activity of these proteins (Anantharaman and Aravind, 2003).


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Fig. 1 Schematic presentation of the NlpC/P60 protein superfamily

The NlpC/P60 superfamily consists of four major protein families. Three of them are represented by bacterial and viral proteins (green letters): the p60-like family, the YaeF-like family, and the AcmB-like family. The fourth group, the LRAT-like protein family, contains eukaryotic (blue letters) and viral 2A proteins (green letter).

The superfamily encompasses four diverse groups of proteins: the P60-like family, the Acm/LytN-like family, the YaeF-like family, and the LRAT-like family (Anantharaman and Aravid; Fig. 1). The P60-like family was typified by the P60 protein of Listeria monocytogenes (Pilgrim et al., 2003), and includes bacterial peptidases with an extracellular location. The NlpC/P60 domain has been demonstrated to be essential for their catalytic activity (Pointing et al., 1999). The Acm/LytN-like family is a very divergent family of proteins typified by its two members, the putative peptidoglycan hydrolase, AcmB (Huard et al., 2003), and a novel cell-wall hydrolase LytN (Sugai et al., 1998). This family is represented by extracellular or membrane proteins functioning mostly as cell-wall hydrolases (Anantharaman and Aravind, 2003). The YaeF-like protein family is typified by the E. coli protein YaeF, and shows a peculiar phylogenetic distribution being present in bacteria and in poxviruses. A function of these proteins is not known, but a similarity with other members of the NlpC/P60 protein superfamily suggests that they might function as proteases (Anantharaman and Aravind, 2003).


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The LRAT family was found only in eukaryotes and animal viruses. The lecithin retinol acyltransferase (LRAT) was identified on the basis of its enzymatic activity, the conversion of all-trans-retinol into retinyl esters, the storage form of retinol (Ruiz et al., 1999). The LRAT ortholog, Egl-26 in C. elegans, has been implicated in vulval development (Wendy and Han, 2002). Other members of the LRAT-like family belong to three subfamilies, the H-REV107-like subfamily containing several tumor suppressors, the subfamily of viral 2A proteins, and a novel, NSE-like protein subfamily (Fig. 1).

Fig. 2 Circular permutation of the NlpC/P60 conservative domain

The NlpC/P60 domain in the Acm/LytN-like and P60-like families has a length of about 60 aminoacids, and following order of the motifs: NCE, GDL, and HWAY. Circular permutation of the domain leads to the rearrangement of the conservative motifs, and distribution through the whole length of a protein. Thus in the LRAT-like proteins the GDL motif is the most amino-terminal, followed the HWAY, and then the NCE motif at the most C-terminus of the protein.
* alternative aminoacid residues presented in several members of the NlpC/P60 protein superfamily


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The NlpC/P60 domain, shared by all four protein families, contains three highly conservative motifs, the NCE, the GDL, and the HWAY motif (Fig. 2). Remarkably, the members of the P60-like, and Acm/LytN-like families have a canonical distribution of these motifs. Namely, the NlpC/P60 domain has a length of about 60 aminoacids, and the following order of the motifs: NCE, GDL, and HWAY. In contrast, a non-canonical distribution of these motifs was demonstrated in the LRAT-like protein family. A circular permutation of the NlpC/P60 domain, and distribution of the conservative motifs through the whole length of the proteins were revealed. The GDL motif became the most amino-terminal, followed the HWAY, and the NCE motifs (Fig. 2; Anantharaman and Aravind, 2003).

Such a circular permutation of the NlpC/P60 catalytic domain, leading to the rearrangement of the GDL motif to the N-terminus, and NCE motif to the C-terminus, supposes different biochemical activities of the members of the NlpC/P60 protein superfamily. It was hypothesised that the genes were acquired by the eukaryotes through lateral transfer of the bacterial precursor. During evolution, the genes underwent drastic changes resulting in a considerable divergence in biochemical functions between the eukaryotic proteins and the bacterial precursors (Anantharaman and Aravind, 2003).

Table 1 Human proteins belonging to the LRAT-like family of proteins

Protein

Identity

Similarity

H-REV107-1/HRASLS3

100%

100%

HRASLS2 (HRAS like suppressor 2)

60%

82%

H-REV107-2/RIG1 (Retinoid inducible gene 1)

51%

66%

HRLP5 (H-rev107 like protein 5)

51%

64%

HRASLS (HRAS like suppressor)

46%

64%

NSE2

31%

46%

Similar to NSE1

31%

46%

NSE1

23%

42%

LRAT (lecithin retinol acyltransferase)

25%

44%

NCBI BLAST search revealed 9 human proteins with a high homology to the H-REV107-1. The first 5 proteins complete the H-REV107-like subfamily, 3 NSE proteins belong to the novel NSE protein subfamily. All non-redundant GenBank CDS and translations+PDB+SwissProt+PIR+PRF databases were used for the search.


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Fig. 3 Aminoacid sequence alignment of the nine human proteins belong to the LRAT-like protein family

Conserved GDL, HWAY, and NCE motifs found in all NlpC/P60 proteins are shown in blue boxes. A transmembrane domain predicted in several proteins indicated in red letters. Proline-rich sequences characteristic for several members of the family only are indicated in green.


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1.2.2.  The LRAT-Like Protein Family

The LRAT-like family consists of eukaryotic proteins, and 2A non-structural proteins of picorna, Aichi, and avian encephalomyelitis (AEV) viruses (Hughes and Stanway, 2000). The function of the viral proteins is not completely elucidated. Several 2A non-structural proteins are trypsin-like or cystein proteases involved in polyprotein processing (Ryan and Flint, 1997), the role of others is unclear.

The human members of the LRAT-like family include 9 homologous proteins: LRAT, 5 proteins belonging to the H-REV107-like subfamily, and 3 proteins forming a novel NSE subfamily (Table 1). The result of an alignment of members of the H-REV107-1, the NSE subfamilies, and LRAT is depicted in the Figure 3.

Thus, proteins of the LRAT-like family share four highly conservative motifs: GDL, HWAY, NCE, and a transmembrane domain at the C-terminus. Additionally, several members contain a prolin-rich region at the N-terminus (Fig. 3, green letters). The function of this region is unknown, although such motifs might be important for protein-protein binding (Kay et al., 2000).

The best-characterised member of the LRAT-like family is the lecithin retinol acyltransferase (LRAT). It is an essential enzyme in vitamin A metabolism mediating the conversion of retinol into retinyl ester (Ruiz et al., 1999). The enzyme is found in those tissues known to be involved in the processing and mobilisation of vitamin A, including the retinal pigment epithelium, the liver, and the intestine. It has been demonstrated that a conserved Cys residue within the NCE motif is essential for LRAT catalysis. The nucleophilic Cys residue reacts with lecithin and becomes acetylated to generate a thiolacyl enzyme intermediate. This fatty acyl fraction reacts then with the vitamin A, and generates retinyl esters (Mondal et al., 2000). Further investigation demonstrated that in addition to the Cys, two His residues, distinguished from the His residues in the HWAY motif are important for the catalysis of LRAT (Mondal et al., 2002).


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The NSE subfamily is a novel subfamily of proteins. These proteins have one aminoacid exchange in the NCE domain: Cys to Ser, resulting in an NSE domain. The NSE2 protein was identified as a protein associated with the plasma membrane in tumor-derived breast cancer cell lines using a proteomics tool. Its potential role in cancer was predicted because of its unique cancer expression profile and identified protein-binding partners which were demonstrated to be implicated in breast cancer tumorigenesis (Adam et al., 2003).

The members of the H-REV107 subfamily are rather poorly characterised, with the exception of the H-REV107-1 and H-REV107-2/TIG3/RIG1 proteins. The HRASLS mouse homologue, Ac1, has been cloned by differential display comparing two mouse cell lines: embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. The gene is expressed in skeletal muscle, heart, brain, and bone marrow in adult mice. It was demonstrated to posses growth inhibitory capacity, and to revert the phenotype of HRAS transformed NIH3T3 cells, proposing that Ac1 can modulate HRAS-mediated signalling pathways (Akiyama et al., 1999).

The H-REV107-2/TIG3/RIG1 is an H-REV107-1 homologous gene (Husmann et al., 1998) which has been identified as a retinoid-responsive gene in primary human keratinocytes (Di Sepio et al., 1998), and as a novel retinoid-inducible gene 1 in human gastric cancer cells (Huang et al., 2000). H-REV107-2/TIG3/RIG1 is a class II tumor suppressor acting as a growth regulator that mediates some of the growth suppressive effects of retinoids. Analysis of truncated forms of this protein demonstrated that the C-terminal hydrophobic domain (Fig. 3) has an important role in determining the intracellular localisation. Both the amino- and carboxy-terminal regions of H-REV107-2/TIG3/RIG1 are required for optimal growth suppression of cells (Deucher et al., 2000). Recently it has been demonstrated that the H-REV107-2/TIG3/RIG1 protein induces apoptosis by negatively regulating extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated kinase (Huang et al., 2002).

Similar to H-REV107-2/TIG3/RIG1, H-REV107-1 belongs to the class II tumor suppressors, and acts as a negative growth modulator (Husmann et al., 1998) by contributing to IFNγ-dependent growth arrest and apoptosis in ovarian carcinoma cells (Sers et al., 2002).


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1.3.  Purpose of this Work

The H-REV107-1 has been identified as a gene down-regulated in RAS-transformed cells (Hajnal et al., 1994). And was shown to suppress cellular growth (Hajnal et al., 1994; Sers et al., 1997). At the begin of this analysis the H-REV107-1 sequence did not provide any clue to the mechanism of its action. No related proteins were found in the databases at this time.

In view of its down-regulation in tumors and tumor cell lines, and its functioning as a growth suppressor, it was decided to study the mechanism of the H-REV107-1 – mediated anti-proliferative effect. To better understand the mechanism of H-REV107-1 cellular function, I performed a yeast two hybrid screening which resulted in the identification of a number of potential interacting partners.

Interaction with these candidates was tested in COS-7 cells using co-immunoprecipitation. A further intention was to determine the H-REV107-1 protein domains responsible for protein-protein interaction. Most importantly, I aimed to define a role for the identified protein-protein interactions in the H-REV107-1 mediated growth suppression and apoptosis.


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