The present invention is directed to compositions involved in cell cycle regulation and methods of use. More particularly, the present invention is directed to genes encoding proteins and proteins involved in cell cycle regulation. Methods of use include use in assays screening for modulators of the cell cycle and use as therapeutics.
Cells cycle through various stages of growth, starting with the M phase, where mitosis and cytoplasmic division (cytokinesis) occurs. The M phase is followed by the G1 phase, in which the cells resume a high rate of biosynthesis and growth. The S phase begins with DNA synthesis, and ends when the DNA content of the nucleus has doubled. The cell then enters G2 phase, which ends when mitosis starts, signaled by the appearance of condensed chromosomes. Terminally differentiated cells are arrested in the G0 phase, and no longer undergo cell division.
The hallmark of a malignant cell is uncontrolled proliferation. This phenotype is acquired through the accumulation of gene mutations, the majority of which promote passage through the cell cycle. Cancer cells ignore growth regulatory signals and remain committed to cell division. Classic oncogenes, such as ras, lead to inappropriate transition from G1 to S phase of the cell cycle, mimicking proliferative extra cellular signals. Cell cycle checkpoint controls ensure faithful replication and segregation of the genome. The loss of cell cycle checkpoint control results in genomic instability, greatly accelerating the accumulation of mutations which drive malignant transformation. Thus, modulating cell cycle checkpoint pathways and other such pathways with therapeutic agents could exploit the differences between normal and tumor cells, both improving the selectivity of radio and chemotherapy, and leading to novel cancer treatments. As another example, it would be useful to control entry into apoptosis.
On the other hand, it is also sometimes desirable to enhance proliferation of cells in a controlled manner. For example, proliferation of cells is useful in wound healing and where growth of tissue is desirable. Thus, identifying modulators which promote, enhance or deter the inhibition of proliferation is desirable.
Despite the desirability of identifying cell cycle components and modulators, there is a deficit in the field of such compounds. Accordingly, it would be advantageous to provide compositions and methods useful in screening for modulators of the cell cycle. It would also be advantageous to provide novel compositions which are involved in the cell cycle.
The present invention provides cell cycle proteins and nucleic acids which encode such proteins. Also provided are methods for screening for a bioactive agent capable of modulating the cell cycle. The method comprises combining a cell cycle protein and a candidate bioactive agent and a cell or a population of cells, and determining the effect on the cell in the presence and absence of the candidate agent. Therapeutics for regulating or modulating the cell cycle are also provided.
In one aspect, a recombinant nucleic acid encoding a cell cycle protein of the present invention comprises a nucleic acid that hybridizes under high stringency conditions to a sequence complementary to that of protein phosphatase type 5 (PP5). In a preferred embodiment, the cell cycle protein provided herein binds to rad9. In another preferred embodiment, PP5 dephosphorylates rad9.
In one embodiment, a recombinant nucleic acid is utilized which comprises a nucleic acid sequence encoding a peptide comprising a binding site for rad9. In another embodiment, a recombinant nucleic acid encoding a cell cycle protein is provided which comprises a nucleic acid sequence having at least 85% sequence identity to a PP5 protein and which binds rad9.
In another aspect of the invention, expression vectors are provided. The expression vectors comprise one or more of the recombinant nucleic acids provided herein operably linked to regulatory sequences recognized by a host cell transformed with the nucleic acid. Further provided herein are host cells comprising the vectors and recombinant nucleic acids provided herein. Moreover, provided herein are processes for producing a cell cycle protein comprising culturing a host cell as described herein under conditions suitable for expression of the cell cycle protein. In one embodiment, the process includes recovering the cell cycle protein.
In another aspect, the present invention provides isolated polypeptides which specifically bind to a cell cycle protein as described herein. Examples of such isolated polypeptides include antibodies. Such an antibody can be a monoclonal antibody. In one embodiment, such an antibody reduces or eliminates the biological function of said cell cycle protein.
Further provided herein are methods for screening for a bioactive agent capable of binding to a cell cycle protein. In one embodiment the method comprises combining a cell cycle protein and a candidate bioactive agent, and determining the binding of said candidate bioactive agent to said cell cycle protein.
In another aspect, provided herein is a method for screening for a bioactive agent capable of interfering with the binding of a cell cycle protein and a rad9 protein. In one embodiment, such a method comprises combining a cell cycle protein a candidate bioactive agent and a rad9 protein, and determining the binding of the cell cycle protein and the rad9 protein. If desired, the cell cycle protein and the rad9 protein can be combined first. In one embodiment, the agent which is identified is a small molecule.
Further provided herein are methods for screening for a bioactive agent capable of modulating the activity of cell cycle protein. In one embodiment the method comprises adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding a cell cycle protein, and determining the effect of the candidate bioactive agent on the cell. In a preferred embodiment, a library of candidate bioactive agents is added to a plurality of cells comprising a recombinant nucleic acid encoding a cell cycle protein.
Also provided herein is a method for screening for a bioactive agent capable of interfering with the dephosphorylation of rad9. In one aspect, the method comprises combining a cell cycle protein PP5 or fragment thereof having phosphatase activity, a candidate bioactive agent and rad9 or a phosphorylated fragment of rad9. The method further comprises determining the dephosphorylation activity of said cell cycle protein on said rad9.
In another aspect of the invention, a method is provided for screening for rad9 variants which modulate PP5 dephosphorylation of rad9. In one embodiment the method comprises combining a cell cycle protein PP5 or fragment thereof having phosphatase activity and a rad9 variant, and determining the dephosphorylation activity of said cell cycle protein on said rad9 variant.
In yet another aspect of the invention a method is provided for screening for PP5 variants which modulate PP5 dephosphorylation of rad9. In one embodiment the method comprises combining a cell cycle protein PP5 variant and rad9 or phosphorylated fragment of rad9, and determining the dephosphorylation activity of said cell cycle protein on said rad9 variant.
Also provided herein is a polypeptide consisting essentially of the carboxy terminal of PP5, wherein said polypeptide has phosphatase activity. Further provided herein is a variant of PP5, wherein said variant differs from a native PP5 polypeptide in that said variant has reduced phosphatase activity. In one embodiment, the reduced phosphatase activity is reduced dephosphorylation of rad9 or a phosphorylated fragment thereof. In a preferred embodiment the variant PP5 binds rad9 but does not have phosphatase activity, or has reduced phosphatase actiavity. In another embodiment, the variant is selected from the group consisting of the polypeptides wherein said native PP5 polypeptide has an H at position 244 and said variant has an A at said position 244, said native PP5 polypeptide has a D at position 274 and an R at position and said variant has an A at said positions 274 and 275, and said native PP5 polypeptide has an N at position 303 and said variant has an A at said position 303. It is understood that homologs of PP5 may-have different numbering of amino acids, and thus in one embodiment, amino acid(s) corresponding to corresponding positions which may not have the exact numbering of the homologs are substituted to form the variants described herein. Also provided herein are nucleic acids encoding the polypeptides and variants provided herein.
Other aspects of the invention will become apparent to the skilled artisan by the following description of the invention.