Cell cycle regulated repressor and DNA element

Details Cell cycle regulated repressor and DNA element Cell cycle regulated repressor and DNA element

1997-09-09

2001-04-10

Schwartzman, Robert A. (Department: 1636)

Chemistry: molecular biology and microbiology

Vector, per se

C536S024100

Reexamination Certificate

active

06214614

ABSTRACT:

The present invention relates to a cell cycle regulated repressor protein which binds to a DNA element present in the control sequences of the human cdc25C gene and other cell cycle regulated genes, as well as the use thereof in cell cycle regulated expression systems.
Eukaryotic and prokaryotic cells replicate by a process of cell division in which the genome of the cell, be it a single molecule as in prokaryotes or a multiplicity of chromosomes as in eukaryotes, is precisely replicated before mitosis. Non-dividing, resting cells are in a phase known as G0. When undergoing division, the cell will move in to G1 phase, usually the longest phase, during which the DNA content is 2n (diploid). This is followed by S phase, in which DNA synthesis takes place and the genome is duplicated. A second G phase follows, G2, in which the cell is in a tetraploid (4n) state. Mitosis (M) then occurs, and the cell reverts to G0/G1. The G2→M transition, which involves cell and nuclear fission, is controlled by a mitosis promoting factor known as cdc2, and a cyclin (cyclin B). The human cdc25C gene encodes a protein phosphatase which activates the cdc2/cyclin B complex prior to the entry into mitosis.
cdc25C mRNA expression is largely restricted to the G2 phase and is developmentally controlled, but the mechanisms of its regulation have not been investigated prior to the present study. In fact, G2-specific transcription has previously not been analysed for any gene in mammalian cells. The molecular mechanisms underlying the periodic induction of genes in the G2 phase are therefore unknown.
cdc25 was originally discovered in
S. pombe
as a cell cycle gene with an essential function in G2→M progression (Russell and Nurse, 1986; for a review see Millar and Russell, 1992). Higher cells contain at least 3 genes with a high degree of similarity to cdc25, termed cdc25A, cdc25B and cdc25C, the latter being the closest kin of the
S. pombe
cdc25 (Millar et al., 1991; Nagata et al., 1991; Sadhu et al., 1990). It is now clear from a number of in vitro studies that the Drosophila, starfish and Xenopus cdc25C genes encode protein phosphatases which presumably directly activate the cdc2/cyclin B complex prior to entry into mitosis (Dunphy and Kumagai, 1991; Gautier et al., 1991; Millar et al., 1991; Sebastian et al., 1993; Strausfeld et al., 1991). In
S. pombe
cdc25 catalysis the dephosphorylation of Tyr-14 in cdc2, thereby reverting the inhibitory action of the wee-1 protein tyrosine kinase (Gould and Nurse, 1989). cdc2 in higher cells is phosphorylated on two residues upon formation of a complex with cyclin B, i.e., Tyr-14 and Thr-15 (reviewed in Millar and Russell, 1992). As in fission yeast, this inactivation of cdc2 is mediated by wee-1, which in mammalian cells has been reported to possess tyrosine and threonine kinase activity (Featherstone and Russell, 1991), and both Tyr-14 and Thr-15 are dephosphorylated by cdc25C (Gautier et al., 1991; Kumagai and Dunphy, 1991; Strausfeld et al., 1991). These observations demonstrate that cdc25 and cdc25C play crucial roles during cell cycle progression in many organisms by triggering the entry into mitosis.
In
S. pombe
, expression of the cdc25 gene is cell cycle-regulated and cdc25 mRNA and protein reach peak levels during G2 (Ducommun et al., 1990; Moreno et al., 1990). This regulation appears to be of particular relevance in view of the fact that the level of cdc25, unlike those of cdc2 or cyclin B, is rate-limiting with respect to entry into M-phase (Ducommun et al., 1990; Edgar and O'Farrell, 1989; Moreno et al., 1990; Russell and Nurse, 1986). In human cells, the level of cdc25C mRNA also increases dramatically in G2, but the abundance of cdc25 protein does not vary greatly during the cell cycle (Millar et al., 1991; Sadhu et al., 1990). The same applies to at least one of the cdc25 forms in
Xenopus oocytes
(Jessus and Beach, 1992).
Many diseases, for example cancer, are associated with aberrant cell proliferation.
Cancer is a disorder of the genetic make-up of somatic cells which results in a clone of cells with an abnormal pattern of growth control. This leads to unrestricted proliferation of the abnormal clone, which may present in the form of a tumour. Available therapy for cancer is based on the premise that cancer cells, being subject to unrestricted growth, undergo more frequent cell division than normal cells. Agents which target dividing cells are therefore seen as useful anti-cancer therapeutics. However, the effectiveness of such agents is limited, since their toxicity to normal cells precludes the administration of sufficiently effective doses. Moreover, within a tumour cell mass, a large proportion of the tissue is not rapidly dividing but is in a resting state. Therefore, even if all the dividing cells are eliminated, the tumour clone is not entirely ablated.
A refinement of such techniques which has been proposed is the use of antibodies or other cell-specific binding agents to target anti-cancer drugs specifically to tumour cells. For example, reference is made to the disclosures of EP 0 590 530 and EP 0 501 215 (Behringwerke AG) and references cited therein. A difficulty with the proposed techniques is that it has proven difficult to selectively target cancer cells over the background of normal tissue cells from which the cancer has developed, since tumour-specific antigens which are targeted by the antibodies or other binding agents are seldom truly tissue-specific.
Recently, gene therapy techniques have been proposed whereby expression systems encoding drugs or enzymes capable of activating prodrugs are targeted to cancer tissue, and preferably expressed selectively in transformed cells. This method allows the introduction of a further level of differentiation between tumour and normal tissues, by exploiting tumour-specific expression vectors as well as tumour-specific targeting systems.
As with antibody delivery systems, however, the drawback with selective expression systems is that background expression levels of the anti-cancer agent encoded by the expression system tend to be excessive, leading to destruction of non-transformed tissue. At the same time, it is difficult to achieve cancer-specific expression using currently available transcription regulation techniques, since qualitatively cancer cells seem to display very few useful differences from the normal tissue from which they derive.
It has now been found that the cdc25C gene and other cell cycle regulated genes, including cyclin A and cdc2 comprise a DNA element which binds a cell cycle specific repressor factor which, when bound, specifically represses transcription of the linked gene.
According to the first aspect of the invention, therefore, there is provided a vector for the expression of a desired gene product in a cell, comprising a structural gene encoding the desired gene product operably linked to a promoter under the control of a DNA repressor element which interacts with a cell cycle specific repressor in order to regulate gene expression in a cell cycle specific manner.
Preferably, the DNA repressor element is derived from a cell cycle regulated gene, such as the cdc25C gene and comprises at least part of the sequence 5′-GCTGGCGGAAGGTTTGAATGG-3′ (SEQ ID NO: 1), or a functionally equivalent mutant or homologue thereof.
Alternatively, the DNA repressor element comprises the sequence 5′-GCTGGCGGAAGGTTTGAATGG-3′ (SEQ ID NO: 1) and a sequence encompassing a transcription initiation site, or any functionally equivalent mutants or homologues thereof.
Preferably, the transcription initiation site is the sequence encompassing the first major transcription initiation site of the cdc25C gene.
The DNA repressor element is preferably derived from the cdc25C gene, the cdc2 gene or the cyclin A gene.
The vector of the invention is a nucleic acid vector which may comprise RNA or DNA. The vector may be of linear or circular configuration and adapted for episomal or integrated existence in the target cell. Vectors based on viruses, such a

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