Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1997-11-25
2001-11-20
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S350000, C530S399000, C514S002600, C514S012200, C514S021800, C514S04400A, C435S069100
Reexamination Certificate
active
06320026
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a polypeptide useful as an anti-tumor agent having the inhibitory activity on the growth of tumor cells and DNA coding for the polypeptide.
BACKGROUND ART
Cell growth is regulated by balance between the growth promoting mechanism and growth inhibitory mechanism. It is considered that at the time of growth, the promoting mechanism is superior to the inhibitory mechanism, and at the time of growth inhibition, the inhibitory mechanism is superior to the promoting mechanism. In normal cells, this balance is maintained suitably and a typical example is a wound-healing process. For example, if epithelial tissues are damaged, the growth promoting mechanism initiates proliferation and migration of cells to compensate for the damaged site, and when the damaged site is filled up, the growth inhibitory mechanism terminates the growth of the cells. In tumor cells, on the other hand, it is considered that the balance in growth control is lost by abnormal activation of the promoting mechanism or by inactivation of the inhibitory mechanism to permit uncontrollable self-growth of cells. By the advance of recent molecular biology and cell biology, molecules involved in controlling the growth of cells have been revealed, but there are not so many reports on the growth inhibitory and arrest mechanisms as compared with studies on the growth promoting mechanism. Recently, studies on the growth inhibitory mechanisms have been conducted extensively from the viewpoint of controlling cell cycle by tumor-suppression genes, G
1
cyclin, cyclin-dependent kinase (CDK), inhibitory factor of CDK etc, but initial stimuli are still not known at the induction of growth arrest.
Known cell growth inhibitory factors having the activity of inhibiting growth of tumor cells include transforming growth factor (TGF)-&bgr; which inhibits the growth of lung cancer cells [Proc. Natl. Acad. Sci. USA, 82, 119-123 (1985)] and epidermal growth factor (EGF) which inhibits the growth of squamous carcinoma cells [Nature, 293, 305-307 (1981)].
It is reported that normal tissues from the cervix of the uterus is placed on a culture plate with their epithelial tissues upward, the epithelial cells initiate to grow spread in the form of a sheet around the tissues after 5 to 7 days in the culture [In Vitro Cell. Div. Biol. Animal, 31, 440-446 (1995)]. This sheet of epithelial cells (referred to hereinafter as “outgrowth”) has the property of expanding concentrically around normal tissues.
It is known that besides known differentiation marker genes, stress inducible genes, tumor marker genes etc., unknown genes are specifically expressed at the time of growth arrest of outgrowth derived from normal epithelial cells in the cervix of the human uterus [Journal of Stomatological Society of Japan, 62, 78-93 (1995)], but it is not known that specifically expressed unknown genes inhibit the growth of tumor cells.
DISCLOSURE OF THE INVENTION
The present invention relates to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (also referred to hereinafter as ETI-1) or a polypeptide consisting of an amino acid sequence wherein one or more amino acids are deleted, replaced or added in the amino acid sequence of SEQ ID NO:1 and having cell growth inhibitory activity (collectively referred to hereinafter as the polypeptide of the present invention), as well as DNA coding for the polypeptide of the present invention, DNA hybridizing with DNA consisting of the nucleotide sequence of SEQ ID NO:1 or 2, or with an oligonucleotide probe prepared based on said nucleotide sequence (collectively referred to hereinafter as the DNA of the present invention). Further, the present invention relates to a recombinant vector comprising the DNA of the present invention (referred to hereinafter as the recombinant vector of the present invention), a transformant obtained by introducing the recombinant vector of the present invention into host cells (referred to hereinafter as the transformant of the present invention), and a process for producing the polypeptide of the present invention comprising culturing the transformant of the present invention, forming and accumulating the polypeptide of the present invention in a medium, and recovering the polypeptide of the present invention from the culture. Furthermore, the present invention relates to a pharmaceutical composition, preferably an anti-tumor agent, comprising the polypeptide of the present invention as an active ingredient. In addition, the present invention relates to a method of preventing or treating tumor comprising administering an effective amount of the polypeptide of the present invention. Finally, the present invention relates to use of the polypeptide of the present invention for producing a pharmaceutical composition useful for preventing or treating tumor.
Besides the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the polypeptide of the present invention may be a polypeptide consisting of said amino acid sequence wherein one or more amino acid residues are deleted, replaced or added in the amino acid sequence of SEQ ID NO:1 as long as having cell growth inhibitory activity. Although the number of deleted, replaced or added amino acid residues is not particularly limited, they are preferably one to dozens amino acid residues, particularly one to a few amino acid residues. In addition, it is preferably a polypeptide consisting of an amino acid sequence wherein one or more amino acids residues are deleted, replaced or added in the amino acid sequence of SEQ ID NO:1 so as to have 50% or more, preferably 70% or more, more preferably 90% or more homology to the amino acid sequence of SEQ ID NO: 5 in homology analysis by BCM search launcher [ALIGN analysis using the algorithm of E. Myers and W. Miller, “Optimal Alignments in Linear Space” (CABIOS, 4, 11-17 (1988))].
The polypeptide consisting of the amino acid sequence wherein one or more amino acids are deleted, replaced or added in the amino acid sequence of SEQ ID NO: 1 and having cell growth inhibitory activity includes those polypeptides of SEQ ID NO: 1 where amino acid residue Glu at the 67-, 70-, 73- or 78-position has been replaced by Ala, or a polypeptide consisting of 92 residues where amino acid residues at the 1- to 89-positions are the same as in SEQ ID NO:l and amino acid residues at the 90 to 92-positions are Lys, Phe and Trp respectively, or a polypeptide consisting of an amino acid sequence as shown in any one of SEQ ID NOS:3 and 5 to 9.
The DNA of the present invention may be any of DNA coding for the polypeptide of the present invention, DNA hybridizing with DNA consisting of a nucleotide sequence as shown in SEQ ID NO: 1 or 2 or with an oligonucleotide probe prepared based on said nucleotide sequence inasmuch as it is DNA coding for a polypeptide having cell growth inhibitory activity.
The DNA of the present invention can be prepared according to the following method.
First, poly(A)
+
RNA is separated from epithelial cells producing the polypeptide of the present invention, e.g. outgrowth cells derived from normal human epithelial cells, and cDNA is prepared from said poly(A)
+
RNA and integrated into a suitable plasmid vector. Then, the resulting recombinant vector is introduced into host cells to prepare a cDNA library, and clones specifically expressed in growth-arrested cells are selected from the resulting cDNA library, and the above DNA is prepared from the resulting clones.
Specifically, a culture plate just outside (about 0.5 mm from) the edge of outgrowth cells of normal human epithelial cells actively growing concentrically is provided with a deep scratch on the bottom of the plate so as to inhibit the expansion of the cell sheet at the site of the scratch. Poly(A)
+
RNA is prepared from the normal human epithelial cells within 7 days, more preferably 2 days after growth was arrested with the scratch made on the culture plate (hereinafter, X days after growth was arres
Masui Tohru
Yamaguchi Satoshi
Carlson Karen Cochrane
Fitzpatrick ,Cella, Harper & Scinto
Japan as represented by Director General of National Institute o
Robinson Patricia
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