Cancer control

Details Cancer control Cancer control

2000-06-23

2003-03-04

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S006120, C435S375000, C435S320100, C435S455000, C514S04400A, C536S024100, C536S024500

Reexamination Certificate

active

06528310

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to cancer control. More specifically, it relates to a method for controlling cancer cell invasion which utilizes control of the matrix metalloproteinase transcription-activating function of E1AF gene, and use thereof in a method for detecting cancer cells, a method for diagnosing cancer invasion and a kit for carrying out these methods.
PRIOR ART
The present inventors succeeded in isolation of E1AF gene encoding E1AF protein, which binds to the enhancer region of E1A gene of adenovirus type 5, from human cell strain HeLa [JP 5-328975 A: Nucleic Acids Research, 21, 547-553 (1993)]. The nucleotide sequence of E1AF gene and the amino acid sequence of E1AF protein expressed by the gene are shown in the Sequence Listing, SEQ ID NO 1, hereinafter.
E1AF gene is a new oncogene belonging to ets group oncogenes (ets family oncogenes) and it has been shown to be human homolog of PEA 3 isolated from mouse [Xin et al., Genes Dev., 6, 481-496 (1992)].
About 30 proteins expressed by ets family oncogenes have been found. They have a common structure called as ETS domain and this structure has DNA-binding activity. It has been considered that proteins expressed by ets family oncogenes function as transcription factors and play important roles in control of gene expression upon cell growth, transformation and the like [Waslylyk et al., Eur. J. Biochem., 211, 7-18 (1993)].
However, the functions of E1AF gene are unclear.
OBJECTS OF THE INVENTION
Objects of the present invention are to clarify the functions of E1AF gene, in particular, the relationship between activation of matrix metalloproteinases and the invasion and metastasis of cancers, and to provide a method for controlling E1AF gene, thereby controlling the invasion and metastasis of cancers, a method for detecting expression of E1AF gene, thereby evaluating malignancy of cancers and a means for carrying out the methods.
SUMMARY OF THE INVENTION
The present inventors have studied the relationship between E1AF gene and matrix metalloproteinases or E1AF gene and the invasion and metastasis of cancers, intensively. As a result, it has been found that E1AF protein expressed by E1AF gene acts on promoters of various matrix metalloproteinase genes to increase their promoter activities, remarkably, thereby enhancing cancer cell invasion. In addition, the present inventors have found that cancer cell invasion can be controlled, if expression of E1AF gene is controlled by using genetic engineering techniques. Furthermore, the present inventors have found that cancer cell invasion can be controlled, if a DNA-binding domain (ETS domain) of E1AF protein is transferred to cancer cells by genetic engineering techniques. Moreover, the present inventors have found methods for detecting cancers and diagnosing cancer invasion by utilizing the expression of E1AF gene as an index. Thus, the present invention has been completed.
That is, briefly, the first aspect of the present invention relates to a method for controlling cancer cell invasion and is characterized by controlling the matrix metalloproteinase-transcription-activating function of E1AF gene, in particular, controlling E1AF or its expression product, or its functional equivalent. The second aspect of the present invention relates to a method for detecting cancers and is characterized by detecting a product expressed by E1AF gene. The third aspect of the present invention relates to a method for diagnosing cancer tissue invasion and is characterized by detecting a product expressed by E1AF gene in cancer tissue isolated from human being. The fourth aspect of the present invention relates to a kit for controlling cancer cell invasion and is characterized by comprising as a constituent component a material for controlling the matrix metalloproteinase transcription-activating function of E1AF gene. The fifth aspect of the present invention relates to a kit for detecting cancers and is characterized by comprising, as a constituent component, a probe which is hybridizable to mRNA of E1AF gene.
DETAILED DESCRIPTION OF THE INVENTION
The wording “functional equivalent” used herein means as follows:
In naturally occurring proteins, in addition to polymorphism or mutation of genes encoding the proteins, mutations such as deletion, substitution, insertion and/or addition of amino acid(s) in their amino acid sequences may occur owing to, for example, modification reactions in a living body and during purification after formation of the proteins. It has been known that there are some proteins which maintain substantially the same physiological and biological activities as those of the original proteins, even after undergoing such mutation. Thus, in case that any significant difference is not found, even when there is a structural difference, it is called a “functional equivalent” herein.
Even when the above mutation is introduced into the amino acid sequence of protein artificially, the situation is unchanged. In this case, many more variants can be produced and, in so far as substantially the same physiological activities are maintained, the variants are recognized to be included in functional equivalents.
For example, in many cases, it is said that methionine residue present in the N-terminal of protein expressed in
E. coli
is removed by the action of methionine aminopeptidase and both products with and without methionine residue are formed according to a particular kind of proteins. However, in many cases, this presence of methionine residue does not influence the activities of proteins. In addition, it has been known that in a polypeptide whose amino acid sequence is similar to human interleukin 2 (IL-2) but wherein a certain cysteine residue has been replaced with serine maintains IL-2 activity [Science, 224, 1431 (1984)].
Furthermore, when proteins are produced by gene engineering techniques, they are often expressed as fusion proteins. For example, an N-terminal peptide chain derived from different protein is added to the N-terminal of the desired protein to increase the amount of the expression of the desired protein; or the desired protein is expressed with addition of a suitable peptide chain to the N- or C-terminal thereof and a carrier having affinity to the peptide chain added thereto is used to facilitate purification of the desired protein.
In addition, it has been known that, for each amino acid, there are 1 to 6 codons which represent the same amino acid on a gene (combination of three nucleotides). Then, many genes encoding one particular amino acid sequence can exist, though it depends on the kind of the amino acid. Genes are by no means present stably in nature and mutation of their nucleotide sequences often occur. There are cases where mutation of a gene does not cause any change in the amino acid sequence encoded by the gene (sometimes called as silent mutation). In such cases, it is considered that different genes encoding the same amino acid sequence are formed. Then, even if a gene encoding a certain amino acid sequence has been isolated, there is a possibility that various kinds of genes encoding the same amino acid sequence are formed during passage of an organism containing the gene.
Moreover, various genes which encode the same amino acid sequence can be produced without any difficulty by utilizing various genetic engineering techniques.
For example, in the production of protein by a genetic engineering technique, when a codon used in an inherent gene encoding the desired protein has a low frequency in a host used, sometimes, only a small amount of the protein is expressed. In such a case, for increasing in the amount of the desired protein expressed, the codon is changed to another one which is frequently used in the host artificially without changing the amino acid sequence to be encoded. Needless to say, in this way, many genes which encode a specific amino acid sequence can be produced artificially. Then, even if nucleotide sequences of genes produced artificially have different nuc

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