Cell division is subdivided into the consecutive phases G.sub.0 or G.sub.1, S, G.sub.2 and M. The S phase is the DNA synthesis phase; it is followed by the transition phase G.sub.2 (G.sub.2 phase), which is followed in turn by the mitosis phase (M phase), in which the parent cell divides into two daughter cells. The resting phase G.sub.0 (G.sub.0 phase) or the transition phase G.sub.1 (G.sub.1 phase) is located between M phase and the S phase.
Cell division is driven forward by a group of protein kinases, i.e. the cyclin/cdk complexes. These comprise a catalytic subunit, known as a cyclin dependent kinase (cdk), for example cdk1, -2, -3, -4, -5, -6, -7 or -8) and a regulatory subunit, i.e. cyclin, for example cyclin A, -B1-B3, -D1-D3, -E, -H, or -C.
Different cdk complexes are particularly active in each phase of the cell cycle. Thus, for example, the cdk complexes cdk4/cyclin D1-3 and cdk6/cyclin D1-3 are active in the mid G.sub.1 phase, the cdk complex cdk2/cyclin E is active in the late G.sub.1 phase, the cdk complex cdk2/cyclin A is active in the S phase, and the cdk complexes cdk1/cyclin B1-3 and cdk1/cyclin A are active in the G.sub.2 /M transition phase.
The cyclin/cdk complexes act by phosphorylating, and consequently activating or inactivating, proteins that are directly or indirectly involved in regulating DNA synthesis and mitosis. In accordance with their function in the cell cycle, the genes for some cyclins and cdk's are periodically transcribed and/or periodically activated or inhibited, for example by controlled degradation of cyclins, by cell cycle phase-specific binding of inhibitors (e.g. p16INK4A, p15INK4B, p21Cip1, P27Kip1, p18INK4C, p19INK4D and P57) or by modification by activating (e.g. cdc25 phosphatases or cdk7/cyclin H) or inhibiting (e.g. weel kinase) enzymes (reviews in Zwicker et al., Progr. Cell Cycle Res., 1:91 (1995); La Thangue, Curr. Opin. Cell Biol., 6:443 (1994); MacLachlan et al., Crit. Rev. Eukaryotic Gene Expr., 5:127 (1995)).
Higher eukaryotes possess at least three cdc25 phosphatases, namely cdc25A, cdc25B and cdc25C. cDNAs encoding these phosphatases already have been cloned and analyzed (Okazaki et al., Gene 178:111 (1996); Galaktionow et al., Cell 67:1181 (1991)). All three phosphatases appear periodically in the cell cycle. However, the activating functions of these cdc25 phosphatases evidently are different (Jinno et al., EMBO J. 13:1549 (1994); Honda et al., FEBS Lett. 318:331 (1993); Hoffmann et al., EMBO J. 13:4302 (1994)).
cdc25A is predominantly expressed in late G.sub.1 phase, and in particular regulates the transition from G.sub.1 to S phase (start of cell cycle) by activating cdk/cyclin complexes; it is itself regulated by Myc (transcription) and Raf (activity). cdc25B dephosphorylates the tyrosines (tyrosine 14 and tyrosine 15) in the ATP-binding pocket of cdk1, thereby leading to their activation; furthermore, it can be stimulated by cyclin B (1-3) independently of cdk1, and its expression is deregulated and augmented in virus (SV40 or HPV)-infected cells. cdc25C also dephosphorylates the tyrosines (tyrosine 14 and tyrosine 15) in the ATP-binding pocket of cdk1, leading to their activation; it is expressed, in particular, in G.sub.2 phase, and regulates entry into M phase.
Periodic expression of cdc25C in the G.sub.2 phase of the cell cycle essentially is regulated by an element (CDE-CHR) in the promoter region of cdc25C; that element is occupied by a repressor protein in G.sub.0 /G.sub.1 phase and is free in G.sub.2 phase. While the nucleotide sequence of this promoter element has been identified and also has been found in the promoters of the cyclin A and cdk1 genes, a somewhat different nucleotide sequence (E2FBS-CHR) has been detected in the promoter for Bmyb. Investigation of the cell cycle-dependent function of these promoter elements has shown that their blockade in the G.sub.0 /G.sub.1 phase is followed by upregulation of the transcription of the relevant gene, where upregulation takes place particularly early (in the mid G.sub.1 phase) for the B-myb gene, in the G.sub.1 /S transition phase for the cyclin A, in the S phase for the cdk1 gene, and only in the late S phase for the cdc25C gene (Zwicker et al., Progress in Cell Cycle Res. 1:91 (1995); Lucibello et al., EMBO J. 14:132 (1995); Liu et al., Nucl. Acids Res. 24:2905 (1995); Zwicker et al., Nucl. Acids Res. 23:3822 (1995); EMBO J. 14:4514 (1995)).
In addition, it has been found that the CDE-CHR element of the promoter for cyclin 25C, cyclin A and the cdk1 gene, and the E2FBS-CHR element of the promoter for the B-myb gene are not only able to inhibit activation and transcription of the homologous genes in the G.sub.0 /G.sub.1 phase, but also are able to inhibit the activation and transcription of other genes. See, for example, WO96/06943, DE19605274.2, DE19617851.7, WO96/06940, WO96/06938, WO96/06941 and WO96/06939.
Those applications disclose preparation of a chimeric promoter, by combining a cell cycle-dependent promoter with a nonspecific, cell-specific, virus-specific or metabolically activatable promoter. This allows for regulated activation of transcription of an effector gene that encodes a protein for the prophylaxis and/or therapy of a disease. Such diseases may, for example, be tumor diseases, leukemias, autoimmune diseases, arthritides, allergies, inflammations, rejection of transplanted organs, diseases of the blood circulatory system or the blood coagulation system, or infections of, or damage to, the central nervous system.
The chimeric promoter is a particular example of this possibility of combining different promoters with a cell cycle-specific promoter element. In the chimeric promoter, the activity of a nonspecific, cell-specific, virus-specific or metabolically activatable activation sequence (or promoter sequence) is to a large extent restricted to the S and G.sub.2 phases of the cell cycle by the CDE-CHR or E2FBS-CHR promoter element which immediately adjoins it downstream.
Subsequent investigations on the mode of function of the CDE-CHR promoter element, in particular, revealed that the cell cycle-dependent regulation by the CDE-CHR element of an upstream activator sequence is to a large extent dependent on whether the activation sequence is activated by transcription factors having glutamine-rich activation domains (Zwicker et al., Nucl. Acids Res. 23:3822 (1995)). Examples of these transcription factors are Sp1 and NF-Y.
As a consequence, this restricts the use of the CDE-CHR promoter element for chimeric promoters. The same must be assumed to be true for the E2F-BS-CHB promoter element of the B-myb gene (Zwicker et al., Nucl. Acids Res. 23:3822 (1995)).
USPTO Backgrounds
USPTO Backgrounds