Gene Ther Mol Biol Vol 1, 629-639. March, 1998.

 

Transcriptional regulation of the H-ras1 proto-oncogene by DNA binding proteins: mechanisms and implications in human tumorigenesis

 

G. Zachos1,2 and D. A. Spandidos1,2

1 Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., Athens 11635; 2 Medical School, University of Crete, Heraklion, Greece.

______________________________________________________________________________________

Correspondence to: Professor D.A. Spandidos, Tel/Fax: +(301)-722 6469

 

Summary

Altered expression of ras genes is a common event in human tumors. Transcriptional regulation of the H-ras1 proto-oncogene occurs through nuclear factors that recognize elements in the promoter region of the gene, in the first and fourth intron and in the VTR unit and involves alternative splicing and specific methylation patterns, as well. Aberrant levels of the Ras p21 protein are detected in a variety of human tumors and are often correlated with clinical and prognostic parameters. Thus, understanding the regulation of the expression of ras genes provides a useful target for gene therapy treatments.

 

 


I. Introduction

ras genes are a ubiquitous eukaryotic gene family. They have been identified in mammals, birds, insects, mollusks, plants, fungi and yeasts. Their sequence is highly conserved, thus revealing the fundamental role they play in cellular proliferation (Spandidos, 1991).

 

A. The structure of ras genes

Three functional ras genes have been identified and characterized in the mammalian genome, H-ras1, K-ras2 and N-ras, as well as two pseudogenes, H-ras2 and K-ras1 (Barbacid, 1987). All three ras genes have a common structure with a 5' non-coding exon (exon -I) and four coding exons (exons I-IV). The introns of the genes differ widely in size and sequence, with the coding sequences of human K-ras spanning more than 35 kb, while those of N-ras and H-ras span approximately 7 and 3 kb, respectively. The K-ras gene has two alternative IV coding exons, thus encoding two proteins, K-RasA and K-RasB (McGrath et al, 1983), with the K-RasB form being more abundant. The H-ras gene also has an alternative exon in the fourth intron (Cohen et al, 1989). In addition, H-ras has a variable tandem repeat sequence (VTR), located downstream of the polyadenylation signal, which exhibits enhancer activity (Spandidos and Holmes, 1987, Cohen et al, 1987).

 

B. Ras proteins: structural characteristics and function

The H-Ras, N-Ras and K-RasA proteins are 189 amino acids long, whereas K-RasB is shorter by one amino acid. They all have a molecular weight of 21kDa and are termed p21 proteins. The p21 proteins are identical at the 86 N-terminal amino acid residues, they possess an 85% homology in the next 80 amino acid residues and diverge highly at the rest of the protein molecule, with the exception of the four C-terminal amino acids which share the common motif CAAX-COOH (C, Cysteine 186; A, Aliphatic amino acid-Leucine, Isoleucine or Valine; X, Methionine or Serine) (Lowy and Willumsen, 1991). The Ras protein is synthesized as pro-p21, undergoes a series of post-translational modifications at the C-terminus increasing the hydrophobicity of the protein and associates with the inner face of the plasma membrane. Sequences at the C-terminus are essential for membrane association and the conserved Cys 186 is required to initiate the post-translational modifications of pro-p21 (Willumsen and Christensen, 1984).

The superfamily of Ras proteins comprises a group of small GTPases, regulating an astonishing diversity of cellular functions (Makara et al, 1996). They are located at the heart of a signal transduction pathway that links cell-surface receptors through a protein kinase cascade to changes in gene expression and cell morphology and to cell division mechanisms. The Ras p21 protein interacts directly with the Raf oncoprotein to recruit the MAP kinases and their subordinates, thus converting a mitogenic signal initiated by membrane receptors with tyrosine kinase activity to a cascade of Serine/ Threonine kinases with multiple targets, including cytoskeleton, transcription factors, inflammatory mediators and other kinases (Avruch et al, 1994, Marshall, 1995).

 

C. ras oncogenes: mechanisms of activation

The ras family of proto-oncogenes, is a frequently detected family of transformation-inducing genes in human tumors. Implication of ras genes in human tumorigenesis occurs by four different mechanisms: point mutations (Kiaris and Spandidos, 1995), gene amplification (Pulciani et al, 1985), insertion of retroviral sequences (Westaway et al, 1986) and alterations in regulation of transcription (Zachos and Spandidos, 1997). With the exception of mutations, all other mechanisms result in activation of the transforming properties of ras genes by quantitative mechanisms.

The c-H-ras1 gene is the best studied member of the family and provides a good example for understanding the mechanisms of gene regulation. The H-ras proto-oncogene expression is regulated by elements located in the promoter region, in intronic sequences and in the 3' end of the gene. In addition, H-ras gene expression is regulated by alternative mechanisms such as DNA methylation and alternative splicing (reviewed by Zachos and Spandidos, 1997). Alterations in the H-ras expression levels are a common mechanism of human tumorigenesis.

 

II. Transcriptional regulation of the H-ras gene from promoter-like sequences

The H-ras gene promoter contains multiple RNA start sites, multiple GC boxes and has no characteristic TATA box (Ishii et al, 1985). These features are characteristic of housekeeping genes. Most promoter region studies have focused on the region upstream of the 5' splice site of the first intron of the gene (nucleotides 1-577), although others consider the SstI fragment (nucleotides 1-1054) that encompasses a part of the first intron as well, to be the gene promoter (Spandidos et al, 1988).

Regulation of gene expression depends on a variety of nuclear factors (Boulikas, 1994). A great number of regulatory elements in the H-ras promoter has been reported, but the results were often controversial, depending on the followed experimental procedure. Transcription factors that interplay on the regulatory regions of the H-ras gene promoter include Sp-1, NF-1, AP-1 and some unknown factors as well.

The Sp-1 is a mammalian DNA binding protein activating transcription by interacting through zinc finger domains with guanine-rich DNA sequences called GC boxes (Berg, 1992). The transcription factor AP-1 is the nuclear factor required to mediate transcription induced by phorbol ester tumor promoters and recognizes a short TGACTCA sequence (Lewin, 1991). Both c-Jun, encoded by members from the jun family (jun, junB, junD), and c-Fos proteins are active components and contribute to the activity of AP-1 by forming c-Jun homodimers as well as c-Jun-c-Fos heterodimers. In addition, there appears to be a mutual antagonism between activation by AP-1 and glucocorticoid receptors at target genes that contain recognition sites for both factors, via protein-protein interactions (Yang-Yen et al, 1990). Finally, the NF-I (CTF) nuclear factor binds the CCAAT element (CAAT box) and is involved in both gene transcription and DNA replication. The NF-I C-terminal region is proline rich and activates transcription through interference with the transcription machinery (Mermod et al, 1989).

Ishii et al (1986), identified six GC boxes that bind the Sp-1 transcription factor as the essential regulatory elements within the H-ras promoter. Using deletion analysis of the H-ras promoter region by focus formation assay in NIH 3T3 cells, Honkawa et al (1987) reported a minimum promoter region of 51 bp length, which was GC rich (78%) and contained a GC box. Lowndes et al (1989) located a 47 bp element, distinct of the one reported by Honkawa et al, that upregulated the transcriptional activity of the promoter region by 20- to 40-fold and contained a GC box and a CCAAT box, binding the NF-1 (CTF) factor. Transient expression assays in which a series of mutants spanning the promoter region of H-ras were ligated to a promoterless chloramphenicol acetyl transferase (CAT) vector, were used in this analysis. Jones et al (1987), also identified two NF-I binding sites, one strong, also noted by Honkawa et al, and one weak. Trimble and Hozumi (1987), using CAT transfection experiments in CV-1 cells, identified a 100 nucleotide region, encompassing the consensus CCAAT box and two Sp-1 sites. However, Nagase et al (1990), using deletion mutants in CAT assays in CV-1 and A-431 cells, suggested that the presence of Sp-1 binding sites at specific positions may not be essential for promoter activity, but a number of Sp-1 binding sites in the region could be required. Lee and Keller (1991), transfected recombinant plasmids encompassing internal deletions and point mutations of the promoter region in HeLa cells and performed CAT assays. They reported a GC box, an unidentified element and a new element CCGGAA directly upstream the GC box, as the most important regulatory elements. Spandidos et al (1988), using recombinant plasmids in CAT activity experiments showed that AP-1-like proteins participate in control of H-ras transcription and identified four TPA responsive-AP-1 binding elements in the H-ras promoter.

A great variety of the transcription initiation sites was also identified (Lowndes et al, 1989, Nagase et al, 1990,

 

 


Figure 1. A synopsis of the reported nuclear factors participating in c-H-ras1 transcriptional regulation. Factors recognize sequences in the H-ras promoter, in intronic sequences and in the VTR region. Exons, rectangles; coding sequences, filled rectangles; VTR, cross-hatched box; B, BamHI cleavage site; S, SstI cleavage site; arrows, major transcriptional initiation sites; ?, unknown regulatory factors; IDX, intron D exon.

 

 

 

 


Lee et al, 1991) using S1 nuclease analysis. A synopsis of the reported nuclear factors that participate on the promoter activity of H-ras, and of the major RNA start sites, are shown in Fig. 1.

 

III. Regulation of the H-ras gene expression from intronic sequences

Intronic sequences play an important role in H-ras regulation. The nuclear factor Sp-1, steroid hormone receptors and the P53 onco-suppressor protein recognize sequences in the first and fourth introns of the H-ras gene.

 

A. The Sp-1 box

There is evidence that the mutant T24 ras 0.8 kb SstI DNA fragment is a more potent activator of gene expression, compared to the corresponding normal H-ras fragment (Spandidos and Pintzas, 1988). A structural basis for this difference was shown to be a 6 bp element in the mutant H-ras fragment, that was absent in the normal H-ras, in the first intron of the gene. This element was proved to contain an Sp-1 binding site (Pintzas and Spandidos, 1991) (Fig. 1).

 

B. The Hormone response elements (HREs)

Steroid hormone receptors produce an enormous number of biological effects in different tissues as hormone activated transcriptional regulators (Beato, 1989, Beato et al, 1995). Such a process requires the coordinate expression of multiple genes and likely candidates are signal transductors, capable of secondarily controlling the transcription of sets of genes that lack steroid hormone response elements. Proto-oncogenes are theoretically well suited for this role, as they exhibit precise temporal patterns of expression during proliferation and differentiation, they participate in signal transduction and regulate the expression of multiple genes in a cascade fashion (Bishop, 1991). Thus, they may integrate signals from steroids and other regulatory factors and amplify the cellular response to the hormone. Evidence for steroid hormone regulation of proto-oncogenes encoding for nuclear transcription factors has already been provided for c-fos, c-jun and c-myc (Hyder et al, 1994).

 

1. Regulation of c-H-ras by steroid hormone receptors

Zachos et al (1995), identified sequences in the 3' end of the first intron and in the fourth intron of the H-ras gene, with high similarity with the glucocorticoid response element (GRE) and estrogen response element (ERE) consensus oligonucleotides, respectively (Fig. 1). Using nuclear extracts from human and murine cell lines in gel retardation assays, it was shown that both glucocorticoid receptor (GR) and estrogen receptor (ER) specifically recognize the corresponding H-ras elements (Zachos et al, 1995).

H-ras p21 protein, is a G-protein involved in signal transduction beginning from transmembrane growth factor and peptide receptors with tyrosine kinase activity (Avruch et al, 1994, Marshall et al, 1995). Steroid receptors, participate in dinstinct signalling mechanisms, involving transcriptional regulation of genes by ligand activated receptors (Beato, 1989, Beato et al, 1995). Thus, hormone regulation of H-ras provides evidence for a direct interaction of these two pathways, allowing the cell to have an additional regulatory "switch" by hormonally modulating the levels of G-proteins at the transcriptional level (Zachos et al, 1996a).

Moreover, the first intron of the gene contains well conserved regions between human and rodents (Hashimoto-Gotoh et al, 1988) and encompasses positive and negative elements influencing H-ras expression, possibly at post-transcriptional level (Hashimoto-Gotoh et al, 1992). It is noteworthy that the GRE, as well as the p53 element that will be discussed later, are located in the conserved region of the intron, thus providing evidence for an essential role in regulating H-ras expression. Moreover, the H-ras GRE is located within the first positive element.

 

2. Interaction of the H-ras and steroid hormone receptors in gynecological cancer

The human endometrium and ovary are major targets for action of glucocorticoids. Sex hormones and steroids act as tumor promoters. The level of receptor binding in H-ras hormone response elements was examined in gel retardation assays, using nuclear extracts from human endometrial and ovarian lesions and from adjacent normal tissue (Zachos et al, 1996b). Increased binding of the glucocorticoid receptor in H-ras GRE was observed in more than 90% of endometrial and in all ovarian tumors tested, compared to the adjacent normal tissue. Moreover, elevated binding of the estrogen receptor in H-ras ERE was found in all pairs of ovarian tumor/ normal tissue examined (Zachos et al, 1996b). Thus, it was proposed that H-ras  is implicated in human gynecological lesions through elevated steroid receptor binding.

In addition, previous data showed cooperation of the H-ras with steroids in cell transformation (Kumar et al, 1990) and overexpression of the Ras p21 in ovarian tumors, compared to normal or benign tumor tissue (Katsaros et al, 1995). By combining these data, it was proposed that the high levels of steroid and sex hormones in human genital tract may result in increased amounts of ligand activated steroid receptors. Furthermore, receptors bind to the H-ras DNA and induce elevated transcription of the H-ras oncogene, resulting in an increased oncogenic potential. Thus, endometrial and ovarian epithelial cells may have a predisposition to develop neoplastic abnormalities in addition to a second tumorigenic event, e.g. viral infection, loss of an onco-suppressor gene, or mutational activation of a proto-oncogene  (Zachos et al, 1996a).

 

C. The p53 element

One of the major roles of wild-type P53 onco-suppressor protein is to trigger cell cycle arrest or apoptosis in response to DNA damage by acting as a sequence specific transcription factor that binds to DNA and activates genes involved in the control of the cell cycle, including p21, gadd45, bax, mdm2 and PCNA (Zambetti et al, 1993, Hainaut, 1995). The mutant forms of P53 promote tumorigenesis by dominant-negative inhibition of wild-type P53 through cross-oligomerization. Moreover, P53 mutants were proved to exert oncogenic functions of their own (Dittmer et al, 1993).

 

1. Regulation of the c-H-ras by the P53 tumor-suppressor protein

H-ras  contains within its first intron sequences that partially match the p53 consensus binding site (Fig. 1). Using gel retardation assays it was shown that wild-type P53, as well as the "hot spot" mutant His 273 recognize the H-ras element with high affinity (Spandidos et al, 1995, Zoumpourlis et al, 1995). Furthermore, the H-ras element functioned as a P53-dependent transcriptional enhancer in the context of a reporter plasmid, thus suggesting that P53 is a physiological regulator of H-ras expression (Spandidos et al, 1995).

Activation of H-ras expression by P53 may seem a paradox, since p53 is a tumor suppressor and H-ras a proto-oncogene. However, precedence has already been established for activation of proto-oncogenes by P53. Wild-type P53 induces expression of mdm-2, whose protein product inhibits the tumor suppressor activities of P53 and Rb (Xiao et al, 1995). Interestingly, there are certain similarities in the organization of the p53 elements of the H-ras and mdm-2 genes, which are not shared with the p53 elements of other genes targeted by P53. In H-ras there are three half sites: two of them are contiguous, while the third is 8 nucleotides upstream. In mdm-2 there are again three half-sites: two are contiguous, while the third one is located 28 nucleotides upstream (Wu et al, 1993). In contrast to H-ras and mdm-2, the elements of the other genes regulated by P53 are contiguous. The organization of the half-sites affects the ability of the P53 protein to recognize these elements. Wild-type P53 reversibly switches between two conformations: the "inactive" T state, with dihedral symmetry, which can recognize only non-contiguous half-sites and the "active" R state, which can recognize even contiguous half-sites (Waterman et al, 1995). Thus, it is suggested that H-ras and mdm-2 genes allow regulation by even the "inactive" T state of P53 protein.

The H-ras p53 element is located within the first intron.  Interestingly, the p53 element of the mdm-2 is also located in the first intron of the gene (Wu et al, 1993). The significance of this is not understood at this time. The mdm-2 has an internal promoter in the first intron. Transcripts initiating at both promoters contain the entire protein sequence, however they differ in the efficiency with which translation is initiated in codon 1. Thus, transcripts that include the first exon mostly express an N-terminally truncated Mdm-2 protein, whereas transcripts from the internal promoter express a full-length protein. P53 induces expression only from the internal promoter and only the full-length form can associate with P53, closing the autoregulatory feedback loop (Barak et al, 1994). It remains to be determined whether P53 induces expression of transcripts initiating at the first intron of H-ras, as well as the biological significance of such transcripts.

The p53 tumor suppressor may therefore exert its cellular effects by coordinate activation of genes that suppress and induce cell proliferation.

 

2. Altered binding of p53 protein to the H-ras element in human tumors

Mutation and overexpression of the p53 tumor suppressor is a common event in human endometrial and ovarian cancer (Berchuck et al, 1994) and is associated with poor prognosis (Levesque et al, 1995, Kihana et al, 1995). Moreover, aberrant regulation of the H-ras gene expression also participates in the development of human gynecological lesions (Zachos and Spandidos, 1997).

Using nuclear extracts from human endometrial and ovarian tumors and from the adjacent normal tissue in gel retardation assays, we examined the binding levels of the P53 protein to the H-ras element (our unpublished results). Elevated P53 binding in the tumor tissue was found in 5/11 (45%) of endometrial and in 2/5 (40%) of ovarian cases. Loss of P53 DNA binding activity was observed in 3/11 (27%) of endometrial and in 1/5 (20%) of ovarian tumors. In the remaining 3/11 (27%) of endometrial and in 2/5 (40%) of ovarian pairs tested, no alteration in the P53 binding levels was observed. In order to interpret the results, all pairs were subsequently tested for mutations in exons 4-9 of the p53 gene using PCR-SSCP analysis. No mutation was observed in any case showing elevated DNA binding activity, thus implying for overexpression of the wild-type p53 gene in these tumors. In addition, no p53 mutational alteration was observed in the cases showing similar DNA binding levels in tumor versus the adjacent normal tissue. However, a mutated allele was detected in all four endometrial and ovarian cases showing loss of P53 DNA binding activity. We therefore suggest that P53 could directly modulate the H-ras oncogenic potential in human endometrial and ovarian lesions, depending on the expressed levels of P53 and the status of the protein (wild-type or mutated forms), thus providing additional evidence for the role of H-ras in human carcinogenesis.

 

IV. The role of the VTR

Variable tandem repeats (VTRs, minisatellites) are highly polymorphic structures characterized by the tandem repetition of short (up to 100 bp) sequence motifs. Several observations on tandemly-repetitive elements within viral genomes (Yates et al, 1984) have led to the speculation that some human minisatellites might serve as regulatory regions for cellular transcription or replication.

 

A. The H-ras minisatellite sequence as transcriptional enhancer

The human H-ras gene contains a VTR region located 1 kb upstream the polyadenylation signal (Fig. 1). It consists of 30 to 100 copies of a 28 bp consensus repeat. Four common alleles and more than 25 rare alleles have been described (Krontiris et al, 1993). It was shown that the H-ras VTR sequences possess endogenous enhancer activity,  independently from orientation, however this activity is promoter specific (Spandidos and Holmes, 1987, Cohen et al, 1987). The 28 bp repeat unit of the minisatellite binds four proteins (p45, p50, p72 and p85) which are members of the rel/ NF-kB family of transcriptional regulatory factors (Trepicchio and Krontiris, 1992).

 

B. VTR rare alleles of the H-ras and ovarian cancer risk

Women who carry a mutation in the BRCA1 gene have an 80% risk of breast cancer and a 40% risk of ovarian cancer by the age of 70 (Easton et al, 1995). The varying penetrance of BRCA1 suggests a role for other genetic and epigenetic factors in tumorigenesis of these individuals. H-ras was the first example of a modifying gene on the penetrance of an inherited cancer syndrome. Rare alleles of the H-ras VTR locus duplicate the magnitude of ovarian cancer risk for BRCA1 carriers, but not the risk for developing breast cancer (Phelan et al, 1996). It was suggested that H-ras VTR alleles show differences in modulating gene transcription, that H-ras VTR alleles are in linkage disequilibrium with other genes important in tumorigenesis, or that rare alleles provide a marker for genomic instability (Phelan et al, 1996).

 

V. The role of the DNA methylation status

DNA methylation is essential for embryonic development and alterations in the DNA methylation status are common in cancer cells. CpG sites in vertebrates are either clustered in 0.5-2 kb regions called


 

Table I. ras gene overexpression in human tumors and correlation with clinical parameters.

 

Tumor type

Frequency

(%)

ras gene

Stage in tumorigenesis

Prognosis of the disease

Neuroblastoma

50-80

H-, ras

early

favourable

Head and neck

54

H-, K-

early

favourable

Esophagus

40

H-

unknown

unknown

Larynx

57-86

H-, K-, N-

unknown

unknown

Thyroid

85

ras

early

unknown

Lung

64-85

ras

late

poor

Liver

60

ras

unknown

unknown

Small intestine

70

ras

unknown

unknown

Stomach

35

K-, ras

late

poor

Pancreas

42

ras

unknown

unknown

Colon

31

H-, K-, ras

early

poor

Breast

65-70

ras

unknown

unknown

Bladder

39-58

H-, K-, N-

early

poor

Endometrium

18-95

ras

late

unknown

Ovary

45

ras

late

poor

Leukemias

39-67

H-, K-, N-

unknown

unknown

 

 


CpG islands, or are dispersed, in which case they are mostly methylated and constitute mutational hotspots (Jones, 1996). The CpG islands are associated with gene promoters (e.g. H-ras) or coding regions (e.g. p16) and are unmethylated in autosomal genes. 5' Methyl-cytosine can affect transcription by altering the DNA binding activities of transcription factors. This could be done either directly, for example binding of trans-acting proteins at AP-2 sites is inhibited (Comb and Goodman, 1990), or indirectly, by enhanced binding of methylated DNA binding protein (MDBP) which stereochemically inhibits DNA binding of transcription factors (Boyes and Bird, 1991).

The promoter region of the H-ras gene is hypomethylated in human tumors compared to the corresponding normal tissue (Feinberg and Vogelstein, 1983). Furthermore, methylation of cis-elements decreases H-ras promoter activity in vitro (Rachal et al, 1989) and inhibits the transforming activity of the oncogene (Borello et al, 1987). It is therefore suggested that epigenetic and reversible mechanisms, like DNA methylation, can regulate the expression of proto-oncogenes and silence genetically activated human oncogenes.

 

VI. Differential expression of the H-ras gene is controlled by alternative splicing

A proportion of H-ras pre-mRNA is spliced to incorporate an alternative exon, termed IDX (intron D exon), which contains an in-frame translational termination codon that prevents expression of the genetic information specified by the exon IV as shown in Fig. 1 (Cohen et al, 1989). The abundance of these transcripts is low, apparently due to message instability or defective processing. The predicted product of the alternate transcript (p19) lacks transforming potential, since the C terminal sequence of p21 that is necessary for attachment of the protein to the inner site of the cellular membrane is absent. It is suggested that alternative splicing patterns operate to suppress the H-ras p21 expression. This negative control is abolished by mutations that interfere with this process.

 

VII. Overexpression of ras genes in human tumors

Overexpression of ras genes is a common event in human tumors (reviewed by Zachos and Spandidos, 1997). Table I summarizes the experimental results by indicating the tumor type where elevated expression of ras genes was observed, the frequency of the overexpression, the activated member of the ras gene family, the stage in tumorigenesis and correlation of altered ras gene expression with prognosis of the disease. Where no particular ras gene is mentioned (referred as: ras), no discrimination between the ras family members was performed, nor was their status (mutated or wild-type alleles)  defined.

Elevated ras gene expression was observed in human neuroblastomas (Spandidos et al, 1992), head and neck tumors (Field, 1991), esophageal (Abdelatif et al, 1991), laryngeal (Kiaris et al, 1995), thyroid (Papadimitriou et al, 1988), lung (Miyamoto et al, 1991), liver (Tiniakos et al, 1989), small intestine (Spandidos et al, 1993), stomach (Motojima et al, 1994), pancreatic (Song et al, 1996), colorectal (Spandidos and Kerr, 1994), breast (Dati et al, 1991), bladder (Ting-jie et al, 1991), endometrial


 

 

 

Figure 2. A synopsis of molecular therapeutic strategies developed against activated ras oncogenes. Strategies include antisense oligonucleotides, ribozymes, farnesyltransferase inhibitors and activation of T-cell response after mutant Ras peptide vaccination of patients.

 

 


 (Long et al, 1988) and ovarian tumors (Scambia et al, 1993) and in leukemias (Gougopoulou et al, 1996). The frequency of elevated expression of the ras  family of genes varies from 30% in endometrial and colorectal tumors, to 85-90% of cases in endometrial, lung and laryngeal tumors. In a number of tumors including neuroblastomas, head and neck, thyroid, colorectal and bladder cancers, ras overexpression is considered to be an early genetic event. However, in lung, stomach, endometrial and ovarian lesions, overexpression of ras genes appears in a later stage in tumorigenesis. Elevated expression of the Ras p21 protein is correlated with poor prognosis in lung, stomach, colorectal, bladder and ovarian lesions, whereas it is a favourable marker for neuroblastomas and head and neck tumors.

 

VIII. Molecular therapeutic strategies

The development of effective molecular strategies for therapy is the aim of tumor biology. Current therapeutic strategies include ribozymes against mutant ras gene products, antisense strategies, inhibitors of Ras protein post-translational modifications and Ras peptide vaccination (Fig. 2).

 

A. Ribozymes

Molecular biology applies the site-specific RNAse properties of ribozymes to gene therapy for cancer. The anti-ras ribozymes are designed to cleave only activated ras RNA (Fig. 2). To develop this strategy into practical means, methods must be developed to accomplish high efficiency delivery of the ribozyme to target neoplastic tissue. An adenoviral-mediated delivery was designed (Feng et al, 1995). Using anti-Ras ribozymes, it was possible to reverse the neoplastic phenotype in mutant H-ras expressing tumor cells with high efficiency (Kashani-Sabet et al, 1994).

 

B. Antisense strategies

The antisense strategy involves reduction of a particular gene expression by introduction of a cDNA segment in antisense orientation, in order to bind the target mRNA and prevent its translation (Stein and Cheng, 1993) (Fig. 2). Critical to the success of such an antisense agent is its ability to enter living cells, to specifically bind the target mRNA and induce RNAse-H cleavage of the target RNA. Activated ras genes, by mutation or overexpression, are a common target of these therapeutic trials in cell-free and in vitro systems (Monia et al, 1992, Schwab et al, 1994).

 

C. Inhibitors of Ras post-translational modifications

Farnesylation of the CAAX motif of Ras protein is essential for the subcellular localization of Ras to the plasma membrane and is critical to Ras cell-transforming activity. Inhibitors of farnesyltransferase have been developed as potential cancer therapeutic agents (Gibbs et al, 1994) (Fig. 2). Requirements for Ras farnesylation inhibitors include specificity for farnesyl protein transferase, ability to inhibit post-translational modifications of the mutant ras specifically, high potency, activity in vivo and lack of toxicity (Kelloff et al, 1997).

 

D. Ras peptide vaccination

Ras peptide vaccination is a recent, developing molecular strategy for cancer therapy. Mutant Ras peptides are candidate vaccines for specific immunotherapy in cancer patients. An amount of mutant Ras p21 is degraded in the cytoplasm and fragments are attached with class I MHC glycoproteins, in the outer surface of the cell membrane (Fig. 2). When vaccinated with a synthetic Ras peptide representing the ras mutation in tumor cells, a transient Ras-specific T-cell response is induced, towards the fragments of mutant Ras protein associated with MHC molecules. Ras peptide vaccination was proved to be effective in 40% of patients with pancreatic cancer (Gjertsen et al, 1995, 1996). However, peptide vaccination of patients, like all other gene therapy strategies previously mentioned, requires considerable development before useful anti-cancer agents can emerge.

 

IX. Concluding remarks

Regulation of the c-H-ras1 gene expression is a complicated procedure, including regulation by a variety of regulatory proteins (transcription factors, hormone receptors, tumor-suppressor proteins), alternative mechanisms (methylation, splicing) and by sequences located in the promoter region, in introns and downstream of the coding sequence. Understanding the molecular mechanisms of the expression of ras genes is of great significance for studying human tumorigenic events and developing effective strategies for gene therapy.

 

 

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