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

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,

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

(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.

References

Abdelatif OMA, Chandler FW, Mills LR, McGuire BS, Pantazis CG and Barret JM (1991) Differential expression of c-myc and H-ras oncogenes in Barret's epithelium-a study using colorimetric in situ hybridazation. Arch Pathol Lab Med 115, 880-885.

Avruch J, Zhang X and Kyriakis JM (1994) Raf meets Ras: completing the framework of a signal transduction pathway. TIBS 19, 279-283.

Barak Y, Gottlieb E, Juven-Gershon T and Oren M (1994) Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential. Genes Dev 8, 1739-1749.

Barbacid M (1987) ras genes. Ann Rev Biochem 56, 779-827.

Beato M, Herrlich P and Schutz G (1995) Steroid hormone receptors: many actors in search of a plot. Cell 83, 851-857.

Beato M (1989) Gene regulation by steroid hormones. Cell 56, 335-344.

Berchuck A, Kohler MF, Marks JR, Wiseman R, Boyd J and Bast RC (1994) The p53 tumor suppressor gene is frequently altered in gynecologic cancers. Am J Obstet Gynecol 170, 246-252.

Berg JM (1992) Sp1 and the subfamily of zinc finger proteins with guanine-rich binding sites. Proc Natl Acad Sci USA 89, 11109-11110.

Bishop JM (1991) Molecular themes in oncogenesis. Cell 64, 235-248.

Borello MG, Pierotti MA, Bongarzone I, Donghi R, Modellini P and Della Porta G (1987) DNA methylation affecting the transforming activity of the human Ha-ras oncogene. Cancer Res 47, 75-79.

Boyes J and Bird A (1991) DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein. Cell 64, 1123-1134.

Boulikas T (1994) A compilation and classification of DNA binding sites for protein transcription factors from vertebrates. Crit Rev Eukaryot Gene Exp 4, 117-321.

Cohen JB, Broz SD and Levinson AD (1989) Expression of the H-ras proto-oncogene is controlled by alternative splicing. Cell 58, 461-472.

Cohen JB, Maureen VW and Levinson AD (1987) A repetitive sequence element 3' of the human c-Ha-ras1 gene has enhancer activity. J Cell Physiol (Suppl) 5: 75-81.

Comb M and Goodman HM (1990) CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2. Nucleic Acids Res 18, 3975-3982.

Dati C, Muraca R, Tazartes O, Antoniotti S, Perrotean I, Giai M, Cortese P, Sismondi P, Saglio G and DeBortoli M (1991) c-ErbB-2 and Ras expression levels in breast cancer are correlated and show a cooperative association with unfavorable clinical outcome. Int J Cancer 47: 833-838.

Dittmer D, Pati S, Zambetti G, Chu S, Teresky AK, Moore M, Finlay C and Levine AJ (1993) Gain of function mutations in p53. Nat Genet 4, 42-46.

Easton D, Ford D and Bishop D (1995) Breast and ovarian cancer incidence in BRCA1 mutation carriers. Am J Hum Genet 56, 265-271.

Feinberg AP and Vogelstein B (1983) Hypomethylation of ras oncogenes in primary human cancers. Biochem Biophys Res Commun 198, 47-54.

Feng M, Cabrera G, Deshane J, Scanlon KJ and Curiel DT (1995) Neoplastic reversion accomplished by high efficiency adenoviral-mediated delivery of an anti-ras ribozyme. Cancer Res 55, 2024-2028.

Field JK (1991) Prognostic implications of ras oncogene expression in head and neck squamous cell carcinoma. In: DA Spandidos, ed. The superfamily of ras related genes: New York, Plenum Press, 213-226.

Gibbs JB, Oliff A and Kohl NE (1994) Farnesyltransferase inhibitors: Ras research yields a potential cancer therapeutic. Cell 77, 175-178.

Gjertsen MK, Bakka A, Breivik J, Saeterdal I, Gadde-Dahl T 3rd, Stokke KT, Salheim BG, Egge TS, Soreide O, Thorsby E and Gaudernack G (1996)Ex vivo ras peptide vaccination in patients with advanced pancreatic cancer: results of a phase I/II study. Int J Cancer 65, 450-453.

Gjertsen MK, Bakka A, Breivik J, Saeterdal I, Solheim BG, Soreide O, Thorsby E and Gaudernack G (1995) Vaccination with mutant ras peptides and induction of T-cell responsiveness in pancreatic carcinoma patients carrying the corresponding RAS mutation. Lancet 346, 1399-1400.

Gougopoulou DM, Kiaris H, Ergazaki M, Anagnostopoulos NI, Grigoraki Y and Spandidos DA (1996) Mutations and expression of the ras family genes in leukemias. Stem Cells 14, 725-729.

Hainaut P (1995) The tumor suppressor protein p53: a receptor to genotoxic stress that controls cell growth and survival. Curr Opin Oncol 7, 76-82.

Hashimoto-Gotoh T, Kikuno R, Takahashi M and Honkawa H (1988) Possible role of the first intron of c-H-ras in gene expression: anti-cancer elements in oncogenes. Anticancer Res 8, 851-860.

Hashimoto-Gotoh T, Takahashi M, Kikuno R, Ishihara H and Tezuka K (1992) Unusual sequence conservation in intron-1 of c-H-ras oncogene and its effect on p21 protein synthesis. In: Spandidos DA, ed. Current Perspectives of Molecular Cellular Oncology. London: JAI Press, 211-230.

Honkawa H, Masahashi W, Hashimoto S, and Hashimoto-Gotoh T (1987) Identification of the principal promoter sequence of the c-Ha-ras transforming oncogene: deletion analysis of the 5' flanking region by focus formation assay. Mol Cell Biol 7, 2933-2940.

Hyder SM, Stancel GM and Loose-Mitchell DS (1994) Steroid hormone-induced expression of oncogene encoded nuclear proteins. Crit Rev Eucaryot Gene Expr 4, 55-116.

Ishii S, Kadonaga JT, Tjian R, Brandy JN, Merlino GT and Pastan I (1986) Binding of the Sp1 transcription factor by the human Harvey ras1 proto-oncogene promoter. Science 232, 1410-1413.

Ishii S, Merlino GT and Pastan I (1985) Promoter region of the human Harvey ras proto-oncogene: Similarity to the EGF receptor proto-oncogene promoter. Science 230, 1378-1381.

Jones PA (1996) DNA methylation errors and cancer. Cancer Res 56, 2463-2466.

Kashani-Sabet M, Funato T, Florenes VA, Fostad O and Scanlon KJ (1994) Suppression of the neoplastic phenotype in vivo by an anti-ras ribozyme. Cancer Res 54, 900-902.

Katsaros D, Theillet C, Zola P, louason G, Sanfilippo B, Isaia E, Arisio R, Giardina G and Sismondi P (1995) Concurrent abnormal expression of erbB-2, myc and ras genes is associated with poor outcome of ovarian cancer patients. Anticancer Res 15, 1501-1510.

Kelloff GJ, Lubet RA, Fay JR, Steele VE, Boone CW, Crowell JA and Sigman CC (1997) Farnesyl protein transferase inhibitors as potential cancer chemopreventives. Cancer Epidemiol Biomarkers Prev 6, 267-282.

Kiaris H and Spandidos DA (1995) Analysis of H-ras, K-ras and N-ras genes for expression, mutation and amplification in laryngeal tumours. Int J Oncol 7, 75-80.

Kiaris H and Spandidos DA (1995) Mutations of ras genes in human tumors (Review). Int J Oncol 7, 413-421.

Kihana T, Hamada K, Inoue Y, Yano N, Iketani H, Murao S, Ukita M and Matsuura S (1995) Mutation and allelic loss of the p53 gene in endometrial carcinoma. Cancer 76, 72-78.

Krontiris TG, Devlin B, Karp DD, Robert NJ and Risch N (1993) An association between the risk of cancer and mutations in the HRAS 1 minisatellite locus. N Eng J Med 329, 517-523.

Kumar R, Sukumar S and Barbacid M (1990) Activation of ras oncogenes preceding the onset of neoplasia. Science 248, 1101-1104.

Lee W and Keller EB (1991) Regulatory elements mediating transcription of the human Ha-ras gene. J Mol Biol 220, 599-611.

Levesque MA, Katsaros D, Yu H, Zola P, Sismondi P, Giardina G and Diamantis EP (1995) Mutant p53 overexpression is associated with poor outcome in patients with well or moderately differentiated ovarian carcinoma. Cancer 75, 1327-1338.

Lewin B (1991) Oncogenic conversion by regulatory changes in transcription factors. Cell 64, 303-312.

Long CA, O'Brien TJ, Sanders MM, Bard DS and Quirk JG Jr (1988) ras oncogene is expressed in adenocarcinoma of the endometrium. Am J Obstet Gynecol 159: 1512-1516.

Lowndes NF, Paul J, Wu J and Allan M (1989) c-Ha-ras gene bidirectional promoter expressed in vitro. Mol Cell Biol 9: 3758-3770.

Lowy DR and Willumsen BM (1993) Function and regulation of ras. Annu Rev Biochem 62, 851-891.

Macara IG, Lounsbury KM, Richards SA, McKiernan C and Bar-Sagi D (1996) The Ras superfamily of GTPases. FASEB J 10, 625-630.

Marshall MS (1995) Ras target proteins in eucaryotic cells. FASEB J 9, 1311-1318.

McGrath JP, Capon DJ, Smith DH, Chen EY, Seeburg PH, Goeddel PV and Levinson AD (1983) Structure and organization of the human Ki-ras proto-oncogene and a related processed pseudogene. Nature 304, 501-506.

Mermod N, O'Neill EA, Kelly TJ and Tjian R (1989) The proline-rich transcriptional activator of CTF/NF-I is dinstict from the replication and DNA binding domain. Cell 58, 741-753.

Miyamoto H, Harada M, Isobe H, Akita HD, Haneda H, Yamaguchi E, Kuzumaki N and Kawakami Y (1991) Prognostic value of nuclear DNA content and expression of the ras oncogene product in lung cancer. Cancer Res 51, 6346-6350.

Monia BP, Johnston JF, Eckers DJ, Zounes MA, Lima WF and Freier SM (1992) Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides. J Biol Chem 267, 19954-19962.

Motojima K, Furui J, Kohara N, Izawa K, Kanematsu T and Shiku H (1994) Expression of Kirsten-ras p21 in gastric-cancer correlates with tumor progression and is prognostic. Diagn Mol Pathol 3, 184-191.

Nagase T, Ueno Y and Ishii S (1990) Transcriptional control of the human Harvey ras proto-oncogene: role of multiple elements in the promoter region. Gene 94, 249-253.

Papadimitriou K, Yiagnisis M, Tolis G and Spandidos DA (1988) Immunohistochemical analysis of the ras oncogene protein in human thyroid neoplasms. Anticancer Res 8, 1223-1228.

Phelan CM, Rebbeck TR, Weber BL, Devilee P, Ruttledge MH, Lynch HT, Lenoir GM, Stratton MR, Easton DF, Ponder BA, Cannon-Albright C, Larsson C, Goldgar DE and Narod SA (1996) Ovarian cancer risk in BRCA1 carriers is modified by the HRAS1 variable number of tandem repeat (VNTR) locus. Nat Genet 12, 309-311.

Pintzas A and Spandidos DA (1991) Sp1 specific binding sites within the human H-ras promoter: potential role of the 6 bp deletion sequence in the T24 H-ras1 gene. Anticancer Res 11: 2067-2070.

Pulciani S, Santos E, Long LK, Sorrentino V and Barbacid M (1985)ras gene amplification and malignant transformation. Mol Cell Biol 5, 2836-2841.

Rachal MJ, Yoo H, Becker FF and Lapeyre J-N (1989) In vitro DNA cytosine methylation of cis-regulatory elements modulates c-Ha-ras promoter activity in vivo. Nucleic Acids Res 17, 5135-5147.

Scambia G, Catozzi L, Panici PB, Ferrandina G, Coronetta F, Barazzi R, Baiocchi G, Uccelli L, Piffanelli A and Mancuso S (1993) Expression of ras oncogene p21 protein in normal and neoplastic ovarian tissues: correlation with histopathologic features and receptors for estrogen, progesterone and epidermal growth factor. Am J Obstet Gynecol 168, 71-78.

Schwab G, Chavany C, Duroux I, Goubin G, Lebeau J, Helene C and Saison-Bechmoaras T (1994) Antisense oligonucleotides adsorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice. Proc Natl Acad Sci USA 91, 10460-10464.

Song MM, Nio Y, Sato Y, Tamura K and Furuse K (1996) Clinicopathological significance of Ki-ras point mutation and p21 expression in benign and malignant exocrine tumors of the human pancreas. Int J Pancreatol 20, 85-93.

Spandidos DA, Arvanitis D and Field JK (1992) Ras p21 expression in neuroblastomas and ganglio-neuroblastomas: correlation with patients' prognosis. Int J Oncol 1, 53-58.

Spandidos DA, Zoumpourlis V, Zachos G, Toas SH and Halazonetis TD (1995) Specific recognition of a transcriptional element within the human H-ras proto-oncogene by the p53 tumor suppressor. Int J Oncol 7, 1029-1034.

Spandidos DA and Holmes L (1987) Transcriptional enhancer activity in the variable tandem repeat DNA sequence downstream of the human Ha-ras1 gene. FEBS Lett 218, 41-46.

Spandidos DA and Kerr IB (1994) Elevated expression of the human ras oncogene family in premalignant and malignant tumours of the colorectum. Br J Cancer 49, 681-688.

Spandidos DA, Liloglou T, Arvanitis D and Gourtsoyiannis NC (1993) ras gene activation in human small intestinal tumors. Int J Oncol 2, 513-518.

Spandidos DA, Nichols RAB, Wilkie NM and Pintzas A (1988) Phorbol ester-responsive H-ras1 gene promoter contains multiple TPA-inducible/ AP-1-binding consensus sequence elements. FEBS Lett 240, 191-195.

Spandidos DA and Pintzas A (1988) Differential potency and trans-activation of normal and mutant T24 human H-ras1 gene promoters. FEBS Lett 232, 269-274.

Spandidos DA (1991) The superfamily of ras-related genes. New York: Plenum Press, 1-338.

Stein CA and Cheng YC (1993) Antisense oligonucleotides as therapeutic agents - is the bullet really magical? Science 261, 1004-1012.

Ting-jie M, Ze W and Nianli S (1991) Correlation between the expression of the p21 ras oncogene product and the biological behavior of bladder tumors. Eur Urol 20, 307-310.

Tiniakos D, Spandidos DA, Kakkanas A, Pintzas A, Pollice L and Tiniakos G (1989) Expression of ras and myc oncogenes in human hepatocellular carcinoma and non-neoplastic liver tissues. Anticancer Res 9, 715-722.

Trepicchio WL and Krontiris TG (1992) Members of the rel/ NF-kB family of transcriptional regulatory proteins bind the HRAS1 minisatellite DNA sequence. Nucleic Acids Res 20, 2427-2434.

Trimble WS and Hozumi N (1987) Deletion analysis of the c-Ha-ras oncogene promoter. FEBS Lett 219, 70-74.

Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ, Drouin J and Karin M (1990) Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62, 1205-1215.

Yates J, Warren, N, Reisman D and Sugden B (1984) A cis-acting element from the Epstein-Barr viral genome that permits stable replication at recombinant plasmids in latently infected cells. Proc Natl Acad Sci USA 81, 3806-3810.

Waterman JLF, Shenk JL and Halazonetis TD (1995) The dihedral symmetry of the p53 tetramerization domain mandates a conformational switch upon DNA binding. EMBO J 14, 512-519.

Westaway D, Parkoff J, Moscovici C and Varmus HE (1986) Identification of a provirally activated c-Ha-ras oncogene in an avian nephroblastoma via a novel procedure: cDNA cloning of a chimaeric viral-host transcript. EMBO J 5, 301-309.

Willumsen BM and Christensen A (1984) The p21 ras C-terminus is required for transformation and membrane association. Nature 310, 583-586.

Wu X, Bayle JH, Olson D and Levine AJ (1993) The p53-mdm2 autoregulatory feedback loop. Genes Dev 7, 1126-1132.

Zachos G and Spandidos DA (1997) Expression of ras proto-oncogenes: regulation and implications in the development of human tumors. Crit Rev Oncol Hematol 26, 65-75.

Zachos G, Zoumpourlis V, Sekeris CE and Spandidos DA (1995) Binding of the glucocorticoid and estrogen receptors to the human H-ras oncogene sequences. Int J Oncol 6, 595-600.

Zachos G, Varras M, Koffa M, Ergazaki M and Spandidos DA (1996a) The association of the H-ras oncogene and steroid hormone receptors in gynecological cancer. J Exp Ther Oncol 1, 335-341.

Zachos G, Varras M, Koffa M, Ergazaki M and Spandidos DA (1996b) Glucocorticoid and estrogen receptors have elevated activity in human endometrial and ovarian tumors as compared to the adjacent normal tissues and recognize sequence elements of the H-ras proto-oncogene. Jpn J Cancer Res 87, 916-922.

Zambetti GP and Levine AJ (1993) A comparison of the biological activities of wild-type and mutant p53. FASEB J 7, 855-865.

Zoumpourlis V, Zachos G, Halazonetis TD, Ergazaki M and Spandidos DA (1995) Binding of wild-type and mutant forms of P53 protein from human tumors to a specific DNA sequence of the first intron of the H-ras oncogene. Int J Oncol 7, 1035-1041.