WO2001036476A2

WO2001036476A2 - Ing2, an iaps associated cell cycle protein, compositions and methods of use

Info

Publication number: WO2001036476A2
Application number: PCT/US2000/031698
Authority: WO
Inventors: Ying Luo; Xiang Xu; Cindy Leo; Betty Huang; Mary Shen
Original assignee: Rigel Pharmaceuticals, Inc.
Priority date: 1999-11-17
Filing date: 2000-11-17
Publication date: 2001-05-25
Also published as: US7420045B1; EP1237916A2; US6737232B1; US7423118B1; EP1237916B1; DE60018605T2; ATE290549T1; AU1660501A; JP2003514548A; WO2001036476A3; CA2390548A1; DE60018605D1

Abstract

The present invention is directed to novel polypeptides, nucleic acids and related molecules which have an effect on or are related to the cell cycle. Also, provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention. Further provided by the present invention are methods for identifying novel compositions which mediate cell cycle bioactivity, and the use of such compositions in diagnosis and treatment of disease.

Description

NOVEL IAPS ASSOCIATED CELL CYCLE PROTEINS, COMPOSITIONS AND METHODS OF USE

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

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 G1 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 pro ferative extracellular 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

SUMMARY OF THE INVENTION

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 set forth in Figure 1 , 3, 5, 7, or 9 In a preferred embodiment, the cell cycle protein provided herein binds to at least one inhibitor of apoptosis proteins (lAPs)

in one embodiment, a recombinant nucleic acid is provided which comprises a nucleic acid sequence as set forth in Figure 1 , 3, 5, 7, or 9 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 sequence as set forth in Figure 1 , 3, 5, 7 or 9 In a further embodiment, provided herein is a recombinant nucleic acid encoding an ammo acid sequence as depicted in Figure 2, 4, 6, 8 or 10

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 cultuπng 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

Also provided herein are recombinant cell cycle proteins encoded by the nucleic acids of the present invention In one aspect, a recombinant polypeptide is provided herein which comprises an ammo acid sequence having at least 80% sequence identity with a sequence as set forth in Figure 2, 4, 6, 8, or 10 In one embodiment, a recombinant cell cycle protein is provided which comprises an ammo acid sequence as set forth in Figure 2, 4, 6, 8, or 10

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 an lAPs In one embodiment, such a method comprises combining a cell cycle protein, a candidate bioactive agent and an lAPs, and determining the binding of the cell cycle protein and the lAPs If desired, the cell cycle protein and the lAPs can be combined first

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

In one embodiment, the present invention provides a method for screening for a bioactive agent capable of modulating the cell cycle which method involves determining the ubiquitin conjugating activity or ubiquitin protein ligase activity of an IAP that is capable of binding to a cell cycle protein

In one embodiment, the present invention provides a method for screening for a bioactive agent capable of modulating the activity of a cell cycle protein which method involves determining the ubiquitin conjugating activity or ubiquitin protein ligase activity of an IAP that is capable of binding to a cell cycle protein

In one embodiment, the present invention provides a method for screening for a bioactive agent capable of modulating apoptosis which method involves determining the ubiquitin conjugating activity or ubiquitin protein ligase activity of an IAP that is capable of binding to a cell cycle protein In one embodiment, the present invention provides a method for screening for a bioactive agent capable of modulating the cell cycle which method involves determining the ubiquitination state of p53 or a component of a p53-contaιnιng protein conglomerate, where p53 can bind to a cell cycle protein, which cell cycle protein can bind to an IAP

In one embodiment, the present invention provides a method for screening for a bioactive agent capable of modulating apoptosis which method involves determining the ubiquitination state of p53 or a component of a p53-contaιnιng protein conglomerate, where p53 can bind to a cell cycle protein, which cell cycle protein can bind to an IAP

In one embodiment, the present invention provides a method for screening for a bioactive agent capable of modulating the activity of a cell cycle protein which method involves determining the ubiquitination state of p53 or a component of a p53-contaιnιng protein conglomerate, where p53 can bind to a cell cycle protein, which cell cycle protein can bind to an IAP

In one embodiment, the present invention provides a method for screening for a bioactive agent capable of modulating the cell cycle which method involves determining the activity of p53 or a p53-contaιnιng protein conglomerate where p53 can bind to a cell cycle protein, which cell cycle protein can bind to an IAP

In one embodiment, the present invention provides a method for screening for a bioactive agent capable of modulating apoptosis which method involves determining the activity of p53 or a p53- containing protein conglomerate, where p53 can bind to a cell cycle protein, which cell cycle protein

In one embodiment, the present invention provides a method for screening for a bioactive agent capable of modulating the activity of a cell cycle protein which method involves determining the activity of p53 or a p53-contaιnιng protein conglomerate, where p53 can bind to a cell cycle protein, which ceil cycle protein can bind to an IAP

Other aspects of the invention will become apparent to the skilled artisan by the following description of the invention

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the nucleic acid sequence of SEQ ID NO 1 , encoding a cell cycle protein ING2, isoform 1

Figure 2 shows the ammo acid sequence of SEQ ID NO 2, which includes the sequence of a cell cycle protein ING2, isoform 1 Figure 3 shows the nucleic acid sequence of SEQ ID NO 3, encoding a cell cycle protein ING2, isoform 2

Figure 4 shows the am o acid sequence of SEQ ID NO 4, which includes the sequence of a cell cycle protein ING2, isoform 2

Figure 5 shows the nucleic acid sequence of SEQ ID NO 5, encoding a cell cycle protein ING2, isoform 3

Figure 6 shows the ammo acid sequence of SEQ ID NO 6, which includes the sequence of a cell cycle protein ING2, isoform 3

Figure 7 shows the nucleic acid sequence of SEQ ID NO 7, encoding a cell cycle protein ING2, isoform 4

Figure 8 shows the ammo acid sequence of SEQ ID NO 8, which includes the sequence of a cell cycle protein ING2, isoform 4

Figure 9 shows the nucleic acid sequence of SEQ ID NO 9, encoding a cell cycle protein ING2, isoform 5

Figure 10 shows the am o acid sequence of SEQ ID NO 10, which includes the sequence of a cell cycle protein ING2, isoform 5

Figure 1 1 shows an alignment of ING2 and ING1 proteins

Figure 12 is a graph depicting p53 activation by ING2

DETAILED DESCRIPTION OF THE INVENTION

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 ceil 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 Other screening assays including binding assays are also provided herein as described below Therapeutics for regulating or modulating the cell cycle are also provided and described herein Diagnostics, as further described below, are also provided herein

A cell cycle protein of the present invention may be identified in several ways "Protein" in this sense includes proteins polypeptides, and peptides The cell cycle proteins of the invention fall into two general classes proteins that are completely novel, i e are not part of a public database as of the time of discovery, although they may have homology to either known proteins or peptides encoded by expressed sequence tags (ESTs) Alternatively, the cell cycle proteins are known proteins, but that were not known to be involved in the cell cycle, i e they are identified herein as having a novel biological function Accordingly, a cell cycle protein may be initially identified by its association with a protein known to be involved in the cell cycle Wherein the cell cycle proteins and nucleic acids are novel, compositions and methods of use are provided herein In the case that the cell cycle proteins and nucleic acids were known but not known to be involved in cell cycle activity as described herein, methods of use, i e functional screens, are provided

In one embodiment provided herein, a cell cycle protein as defined herein has one or more of the following characteristics binding to at least one IAP, homology with a p33lNG1 protein, and cell cycle protein activity as described herein The homology to such p33INGF1 proteins can be found as described below In one embodiment, homology is found using the following database and parameters Database Non-redundant GenBank CDS translations+PDB+SwissProt+SPupdate+PIR, Lambda of 0 316, K of 0 133 and H of 0, Gapped Lambda of 0 27, K of 0 047, and H of 4 94e-324, Matrix is BLOSUM62, Gap Penaiities Existence 11 , Extension 1

In one embodiment, the cell cycle protein is termed ING2 herein In a preferred embodiment, ING2 includes any one of the isoforms shown in the figures The characteristics described below can apply to any of the cell cycle proteins provided herein, however, ING2 is used for illustrative purposes ING2 has similarity to proteins belonging to a family of tumor suppressors Proper growth inhibition or apoptotic regulation, facilitates cell death and tumor suppression Preferably, ING2 binds to at least one lAPs Studies have reported on lAPs, for example, one study reports that introduction of lAPs (clAPs), clAP1 and clAP2, to the apoptotic genes (reaper and grim) demonstrated physical interaction and frustration of apoptosis McCarthy and Dixit, J Biol Chem , 273(37) 24009-15 (1998) Reaper and grim participate in apoptosis function by irreversibly blocking voltage-gated K+ channels thereby inducing cell death Avdonin, et al , PNAS USA, 95(20) 11703-8 (1998) lAPs are also reported to interact with HID Vucic, et al , Mol Cell Biol , 18(6) 3300-9 (1998) Also regarding lAPs, see, for example, Vucic, PNAS USA, 94(19) 10183-8 (1997), Hay, et al , Cell, 83(7) 1253-62 (1995)

Moreover, in a preferred embodiment, ING2 activates p53 binding site controlled promoters in the presence or absence of p53 In a more preferred embodiment, ING2 activation is synergistic with p53 Most preferably, ING2 suppresses tumor growth, preferably in the presence of p53

ING1 family members, to which ING2 shares some homology, are tumor suppressors The novel cell cycle proteins provided herein share greater homology with the ING2 sequences in the figures than do ING1 family members as described herein, or other known proteins A study reports that the ING1 gene encodes p33ING1 , a nuclear protein The properties of ING1 point to several regulatory functions of the cell cycle ING1 may be linked to negative regulation of cell proliferation, the control of cellular aging, anchorage dependence and apoptosis Garkavtsev, et al , Nature, 391 (6664) 295-8 (1998) The function of ING1 may depend upon the activity of p53, a tumor-suppression gene It is postulated that the proteins encoded for growth inhibition and tumor suppression via ING1 and p53 are interrelated and depend upon the activity of each other Further, transcription activation of p53 has shown to depend upon the expression of ING1 , immunoprecipitation studies indicate a physical association between p33ING1 and proteins encoded by p53 Regarding ING1 genes and proteins, also see, e g , Helbing, et al , Cancer Res , 57(7) 1255-8 (1997), Garkavtsev, et al , Nat Genet , 14(4) 415-20 (1996), Shimada, et al , Cytogenet Cell Genet , 83(3-4) 232-5 (1998)

in one embodiment, cell cycle nucleic acids or cell cycle proteins can be initially identified by substantial nucleic acid and/or ammo acid sequence identity or similarity to the sequence(s) provided herein In a preferred embodiment, cell cycle nucleic acids or cell cycle proteins have sequence identity or similarity to the sequences provided herein as described below and one or more of the cell cycle protein bioactivities as further described below Such sequence identity or similarity can be based upon the overall nucleic acid or ammo acid sequence

In a preferred embodiment, a protein is a "cell cycle protein" as defined herein if the overall sequence identity of the amino acid sequence of Figure 2, 4, 6, 8, or 10 is preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90% In some embodiments the sequence identity will be as high as about 93 to 95 or 98%

In another preferred embodiment, a cell cycle protein has an overall sequence similarity with the am o acid sequence of Figure 2, 4, 6, 8, or 10 of greater than about 80%, more preferably greater than about 85%, even more preferably greater than about 90% and most preferably greater than 93% In some embodiments the sequence identity will be as high as about 95 to 98 or 99%

As is known in the art, a number of different programs can be used to identify whether a protein (or nucleic acid as discussed below) has sequence identity or similarity to a known sequence Sequence identity and/or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv Appl Math 2 482 (1981 ), by the sequence identity alignment algorithm of Needleman & Wunsch, J Mol Biool 48 443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wl), the Best Fit sequence program described by Devereux et al , Nucl Acid Res 12387-395 (1984), preferably using the default settings, or by inspection Preferably, percent identity is calculated bv FastDB based upon the following parameters mismatch penalty of 1 , gap penalty of 1 , gap size penalty of 0 33, and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R Liss, Inc

An example of a useful algorithm is PILEUP PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments It can also plot a tree showing the clustering relationships used to create the alignment PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J Mol Evol 35 351-360 (1987), the method is similar to that described by Higgins & Sharp CABIOS 5 151 -153 (1989) Useful PILEUP parameters including a default gap weight of 3 00, a default gap length weight of 0 10, and weighted end gaps

Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al , J Mol Biol 215, 403-410, (1990) and Karlin et al , PNAS USA 90 5873-5787 (1993) A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al , Methods in Enzymology, 266 460-480 (1996), http //blast wustl/edu/blast/ README html] WU-BLAST-2 uses several search parameters, most of which are set to the default values The adjustable parameters are set with the following values overlap span =1 , overlap fraction = 0 125, word threshold (T) = 11 The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched, however, the values may be adjusted to increase sensitivity

An additional useful algorithm is gapped BLAST as reported by Altschul ef al Nucleic Acids Res 25 3389-3402 Gapped BLAST uses BLOSUM-62 substitution scores, threshold T parameter set to 9, the two-hit method to trigger ungapped extensions, charges gap lengths of k a cost of \ 0+k, X_u set to 16, and X_g set to 40 for database search stage and to 67 for the output stage of the algorithms Gapped alignments are triggered by a score corresponding to -22 bits

A % ammo acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored)

In a similar manner, "percent (%) nucleic acid sequence identity" with respect to the coding sequence of the polypeptides identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the cell cycle protein A preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0 125, respectively The alignment may include the introduction of gaps in the sequences to be aligned In addition, for sequences which contain either more or fewer ammo acids than the protein encoded by the sequences in the Figures, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical ammo acids in relation to the total number of ammo acids Thus, for example, sequence identity of sequences shorter than that shown in the Figure, as discussed below, will be determined using the number of ammo acids in the shorter sequence, in one embodiment In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, efc

In one embodiment, only identities are scored positively (+1 ) and all forms of sequence variation including gaps are assigned a value of "0", which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations Percent sequence identity can be calculated, for example, by dividing the numDer of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100 The "longer" sequence is the one having the most actual residues in the aligned region

As will be appreciated by those skilled in the art the sequences of the present invention may contain sequencing errors That is, there may be incorrect nucleosides, frameshifts, unknown nucleosides, or other types of sequencing errors in any of the sequences; however, the correct sequences will fall within the homology and stringency definitions herein

Cell cycle proteins of the present invention may oe shorter or longer than the ammo acid sequence encoded by the nucleic acid shown in the Figure Thus, in a preferred embodiment, included within the definition of cell cycle proteins are portions or fragments of the am o acid sequence encoded by the nucleic acid sequence provided herein In one embodiment herein, fragments of cell cycle proteins are considered cell cycle proteins if a) tney share at least one antigenic epitope, b) have at least the indicated sequence identity, c) and preferably have cell cycle biological activity as further defined herein In some cases, where the sequence is used diagnostically, that is, when the presence or absence of cell cycle protein nucleic acid is determined, only the indicated sequence identity is required The nucleic acids of the present invention may also be shorter or longer than the sequence in the Figure The nucleic acid fragments include any portion of the nucleic acids provided herein which have a sequence not exactly previously identified, fragments having sequences with the indicated sequence identity to that portion not previously identified are provided in an embodiment herein

In addition, as is more fully outlined below, cell cycle proteins can be made that are longer than those depicted in the Figure, for example, by the addition of epitope or purification tags, the addition of other fusion sequences, or the elucidation of additional coding and non-coding sequences As described below, the fusion of a cell cycle peptide to a fluorescent peptide, such as Green Fluorescent Peptide (GFP), is particularly preferred

Cell cycle proteins may also be identified as encoded by cell cycle nucleic acids which hybridize to the sequence depicted in the Figure, or the complement thereof, as outlined herein Hybridization conditions are further described below

In a preferred embodiment, when a cell cycle protein is to be used to generate antibodies, a cell cycle protein must share at least one epitope or determinant with the full length protein By "epitope" or "determinant" herein is meant a portion of a protein which will generate and/or bind an antibody Thus, in most instances, antibodies made to a smaller cell cycle protein will be able to bind to the full length protein In a preferred embodiment, the epitope is unique, that is, antibodies generated to a unique epitope show little or no cross-reactivity The term "antibody" includes antibody fragments, as are known in the art, including Fab Fab₂, single chain antibodies (Fv for example), chimeπc antibodies, etc , either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies

In a preferred embodiment, the antibodies to a cell cycle protein are capable of reducing or eliminating the biological function of the cell cycle proteins described herein, as is described below That is, the addition of anti-cell cycle protein antibodies (either polyclonal or preferably monoclonal) to cell cycle proteins (or cells containing cell cycle proteins) may reduce or eliminate the cell cycle activity Generally, at least a 25% decrease in activity is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred

The cell cycle antibodies of the invention specifically bind to cell cycle proteins In a preferred embodiment, the antibodies specifically bind to cell cycle proteins By "specifically bind" herein is meant that the antibodies bind to the protein with a binding constant in the range of at least 10^"4- 10⁶ M ¹ , with a preferred range being 10 ⁷ - 10⁹ M ¹ Antibodies are further described beiow

In the case of the nucleic acid, the overall sequence identity of the nucleic acid sequence is commensurate with am o acid sequence identity but takes into account the degeneracy in the genetic code and codon bias of different organisms Accordingly, the nucleic acid sequence identity may be either lower or higher than that of the protein sequence Thus the sequence identity of the nucleic acid sequence as compared to the nucleic acid sequence of the Figures is preferably greater than 75%, more preferably greater than about 80%, particularly greater than about 85% and most preferably greater than 90% In some embodiments the sequence identity will be as high as about 93 to 95 or 98%

In a preferred embodiment, a cell cycle nucleic acid encodes a cell cycle protein As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the cell cycle proteins of the present invention Thus, having identified a particular am o acid sequence, those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the ammo acid sequence of the cell cycle protein

In one embodiment, the nucleic acid is determined through hybridization studies Thus, for example, nucleic acids which hybridize under high stringency to the nucleic acid sequence shown in the Figure, or its complement is considered a cell cycle nucleic acid High stringency conditions are known in the art, see for example Maniatis et al , Molecular Cloning A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed Ausubel, et al , both of which are hereby incorporated by reference Stringent conditions are sequence-dependent and will be different in different circumstances Longer sequences hybridize specifically at higher temperatures An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993) Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (TJ for the specific sequence at a defined ionic strength pH The T_m is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium) Stringent conditions will be those in which the salt concentration is less than about 1 0 sodium ion, typically about 0 01 to 1 0 M sodium ion concentration (or other salts) at pH 7 0 to 8 3 and the temperature is at least about 30^°C for short probes (e g 10 to 50 nucleotides) and at least about 60°C for long probes (e g greater than 50 nucleotides) Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide

In another embodiment less stringent hybridization conditions are used, for example, moderate or low stringency conditions may be used, as are known in the art, see Maniatis and Ausubel, supra, and Tijssen, supra

The cell cycle proteins and nucleic acids of the present invention are preferably recombinant As used herein and further defined below, "nucleic acid" may refer to either DNA or RNA, or molecules which contain both deoxy- and nbonucleotides The nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids Such nucleic acids may also contain modifications in the πbose-phosphate backbone to increase stability and half life of such molecules in physiological environments

The nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence As will be appreciated by those in the art, the depiction of a single strand ("Watson") also defines the sequence of the other strand ( 'Crick"), thus the sequences depicted in the Figures also include the complement of the sequence By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature Thus an isolated cell cycle nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i e using the vivo cellular machinery of the host cell rather than in vitro manipulations, however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention

Similarly, a "recombinant protein" is a protein made using recombinant techniques, i e through the expression of a recombinant nucleic acid as depicted above A recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure For example, an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0 5%, more preferably at least about 5% by weight of the total protein in a given sample A substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred The definition includes the production of a cell cycle protein from one organism in a different organism or host cell

Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a mducible promoter or high expression promoter, such that the protein is made at increased concentration levels Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or am o acid substitutions, insertions and deletions, as discussed below

In one embodiment, the present invention provides cell cycle protein variants These variants fall into one or more of three classes substitutional, insertional or deletional variants These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding a cell cycle protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant and thereafter expressing the DNA in recombinant cell culture as outlined above However variant cell cycle protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques Ammo acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or mterspecies variation of the cell cycle protein am o acid sequence The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below While the site or region for introducing an am o acid sequence variation is predetermined, the mutation per se need not be predetermined For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed cell cycle variants screened for the optimal combination of desired activity Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of cell cycle protein activities

Ammo acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 ammo acids, although considerably larger insertions may be tolerated Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger

Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative Generally these changes are done on a few am o acids to minimize the alteration of the molecule However, larger changes may be tolerated in certain circumstances When small alterations in the characteristics of the cell cycle protein are desired, substitutions are generally made in accordance with the following chart

Chart I Original Residue Exemplary Substitutions

Ala Ser Arg Lys

Asp Glu

Cys Ser

Gin Asn Glu Asp

Gly Pro

His Asn, Gin lie Leu, Val

Leu lie, Val Lys Arg, Gin, Glu

Met Leu, lie

Phe Met, Leu, Tyr

Ser Thr

Thr Ser Trp Tyr

Tyr Trp, Phe

Val lie, Leu Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Chart I For example, substitutions may be made which more significantly affect the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophihc residue, e g seryl or threonyl, is substituted for (or by) a hydrophobic residue, e g leucyl, isoleucyl, phenylalanyl, valyl or alanyl, (b) a cysteine or proline is substituted for (or by) any other residue, (c) a residue having an electropositive side chain, e g lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e g glutamyl or aspartyl, or (d) a residue having a bulky side chain, e g phenylalamne, is substituted for (or by) one not having a side chain, e g glycine

The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the cell cycle proteins as needed Alternatively, the variant may be designed such that the biological activity of the cell cycle protein is altered For example, glycosylation sites may be altered or removed

Covalent modifications of cell cycle polypeptides are included within the scope of this invention One type of covalent modification includes reacting targeted ammo acid residues of a cell cycle polypeptide with an organic deπvatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of a cell cycle polypeptide Derivatization with bifunctional agents is useful, for instance, for crosslmking cell cycle to a water-insoluble support matrix or surface for use in the method for purifying anti-cell cycle antibodies or screening assays, as is more fully described below Commonly used crosslmking agents include, e g , 1 ,1 -bιs(dιazoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccmimide esters, for example, esters with 4-azιdo- salicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dιthιobιs- (succinimidylpropionate), bifunctional maleimides such as bιs-N-maleιmιdo-1 ,8-octane and agents such as methyl-3-[(p-azιdophenyl)dιthιo]propιoιmιdate

Other modifications include deamidation of glutaminyl and asparagmyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the "-ammo groups of lysine, arginine, and histidine side chains [T E Creighton, Proteins Structure and Molecular Properties, W H Freeman & Co , San Francisco, pp 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group

Another type of covalent modification of the cell cycle polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence cell cycle polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence cell cycle polypeptide

Addition of glycosylation sites to cell cycle polypeptides may be accomplished by altering the ammo acid sequence thereof The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence cell cycle polypeptide (for O- nked glycosylation sites) The cell cycle ammo acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the cell cycle polypeptide at preselected bases such that codons are generated that will translate into the desired am o acids

Another means of increasing the number of carbohydrate moieties on the cell cycle polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide Such methods are described in the art, e g , in WO 87/05330 published 11 September 1987, and in Aplin and Wπston, CRC Cπt Rev Biochem , pp 259-306 (1981 )

Removal of carbohydrate moieties present on the cell cycle polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for ammo acid residues that serve as targets for glycosylation Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al , Arch Biochem Biophys , 259 52 (1987) and by Edge et al , Anal Biochem , 118 131 (1981 ) Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al , Meth Enzymol , 138 350 (1987)

Another type of covalent modification of cell cycle comprises linking the cell cycle polypeptide to one of a variety of nonproteinaceous polymers, e g , polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U S Patent Nos 4 640,835, 4,496,689, 4,301 ,144, 4,670,417, 4,791 ,192 or 4,179,337

Cell cycle polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising a cell cycle polypeptide fused to another, heterologous polypeptide or ammo acid sequence In one embodiment, such a chimeric molecule comprises a fusion of a cell cycle polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind The epitope tag is generally placed at the ammo-or carboxyl-terminus of the cell cycle polypeptide The presence of such epitope-tagged forms of a cell cycle polypeptide can be detected using an antibody against the tag polypeptide Also, provision of the epitope tag enables the cell cycle polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag In an alternative embodiment, the chimeric molecule may comprise a fusion of a cell cycle polypeptide with an immunoglobulm or a particular region of an immunoglobulm For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule as discussed further below Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidme (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8.2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5 3610-3616 (1985)]; and the Herpes Simplex virus glycoprotem D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnoloqy, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol Chem., 266:15163-15166 (1991 )]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc. Natl. Acad Sci USA, 87 6393-6397 (1990)1.

In an embodiment herein, cell cycle proteins of the cell cycle family and cell cycle proteins from other organisms are cloned and expressed as outlined below. Thus, probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related cell cycle proteins from humans or other organisms As will be appreciated by those in the art, particularly useful probe and/or PCR primer sequences include the unique areas of the cell cycle nucleic acid sequence. As is generally known in the art, preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosme as needed. The conditions for the PCR reaction are well known in the art. It is therefore also understood that provided along with the sequences in the sequences listed herein are portions of those sequences, wherein unique portions of 15 nucleotides or more are particularly preferred. The skilled artisan can routinely synthesize or cut a nucleotide sequence to the desired length.

Once isolated from its natural source, e.g , contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant cell cycle nucleic acid can be further- used as a probe to identify and isolate other cell cycle nucleic acids It can also be used as a "precursor" nucleic acid to make modified or variant cell cycle nucleic acids and proteins

Using the nucleic acids of the present invention which encode a cell cycle protein, a variety of expression vectors are made The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcπptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the cell cycle protein. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a πbosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotem that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a πbosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation As another example, operably linked refers to DNA sequences linked so as to be contiguous, and in the case of a secretory leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the cell cycle protein, for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the cell cycle protein in Bacillus Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells

In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, nbosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences

Promoter sequences encode either constitutive or inducible promoters The promoters may be either naturally occurring promoters or hybrid promoters Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention

In addition, the expression vector may comprise additional elements For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector Constructs for integrating vectors are well known in the art

In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells Selection genes are well known in the art and will vary with the host cell used

A preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference

Cell cycle proteins of the present invention are produced by cultuπng a host cell transformed with an expression vector containing nucleic acid encoding a cell cycle protein, under the appropriate conditions to induce or cause expression of the cell cycle protein The conditions appropriate for cell cycle protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction In addition, in some embodiments, the timing of the harvest is important For example, the baculoviral systems used in insect cell expression are Iytic viruses, and thus harvest time selection can be crucial for product yield

Appropriate host cells include yeast, bacteria, archebacteπa, fungi, and insect and animal cells, including mammalian cells Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid and iymphoid cell lines

In a preferred embodiment, the cell cycle proteins are expressed in mammalian cells Mammalian expression systems are also known in the art, and include retroviral systems A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for cell cycle protein into mRNA A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site A mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter

Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence The 3' terminus of the mature mRNA is formed by site- specific post-translational cleavage and polyadenylation Examples of transcription terminator and polyadenlytion signals include those derived form SV40 The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotιde(s) in hposomes, and direct microinjection of the DNA into nuclei

In a preferred embodiment, cell cycle proteins are expressed in bacterial systems Bacterial expression systems are well known in the art

A suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of cell cycle protein into mRNA A bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan Promoters from bacteriophage may also be used and are known in the art In addition, synthetic promoters and hybrid promoters are also useful, for example, the tac promoter is a hybrid of the trp and lac promoter sequences Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription

In addition to a functioning promoter sequence, an efficient πbosome binding site is desirable In E coli, the nbosome binding site is called the Shine-Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 1 1 nucleotides upstream of the initiation codon

The expression vector may also include a signal peptide sequence that provides for secretion of the ceil cycle protein in bacteria The signal sequence typically encodes a signal peptide comprised of hydrophobic ammo acids which direct the secretion of the protein from the cell, as is well known in the art The protein is either secreted into the growth media (gram-positive bacteria) or into the peπplasmic space, located between the inner and outer membrane of the cell (gram- negative bacteria)

The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycm, neomycin and tetracyc ne Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways These components are assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others.

The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.

In one embodiment, cell cycle proteins are produced in insect cells Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art

In a preferred embodiment, cell cycle protein is produced in yeast cells Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha. Kluyveromyces fragilis and K. lactis, Pichia guillenmondii and P. pastoπs, Schizosaccharomyces pombe, and Yarrowia lipolytica Preferred promoter sequences for expression in yeast include the inducible GAL1 ,10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokmase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexok ase, phosphofructokinase, 3- phosphoglycerate mutase, pyruvate kmase, and the acid phosphatase gene Yeast selectable markers include ADE2, HIS4, LEU2, TRP1 , and ALG7, which confers resistance to tunicamycin; the neomycm phosphotransferase gene, which confers resistance to G418, and the CUP1 gene, which allows yeast to grow in the presence of copper ions.

The cell cycie protein may also be made as a fusion protein, using techniques well known in the art. Thus, for example, for the creation of monoclonal antibodies, if the desired epitope is small, the cell cycle protein may be fused to a carrier protein to form an immunogen Alternatively, the cell cycle protein may be made as a fusion protein to increase expression, or for other reasons For example, when the cell cycle protein is a cell cycle peptide, the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes Similarly, cell cycle proteins of the invention can be linked to protein labels, such as green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), etc.

In one embodiment, the cell cycle nucleic acids, proteins and antibodies of the invention are labeled. By "labeled" herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes, b) immune labels, which may be antibodies or antigens, and c) colored or fluorescent dyes The labels may be incorporated into the compound at any position

In a preferred embodiment, the ceil cycle protein is purified or isolated after expression. Cell cycle proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusmg For example, the cell cycle protein may be purified using a standard anti-cell cycle antibody column Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful For general guidance in suitable purification techniques, see Scopes, R , Protein Purification, Spπnger-Verlag, NY (1982) The degree of purification necessary will vary depending on the use of the cell cycle protein In some instances no purification will be necessary

Once expressed and purified if necessary, the cell cycle proteins and nucleic acids are useful in a number of applications

The nucleotide sequences (or their complement) encoding cell cycle proteins have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA Cell cycle protein nucleic acid will also be useful for the preparation of cell cycle proteins by the recombinant techniques described herein

The full-length native sequence cell cycle protein gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate other genes (for instance, those encoding naturally-occurring variants of cell cycle protein or cell cycle protein from other species) which have a desired sequence identity to the cell cycle protein coding sequence Optionally, the length of the probes will be about 20 to about 50 bases The hybridization probes may be derived from the nucleotide sequences herein or from genomic sequences including promoters, enhancer elements and mtrons of native sequences as provided herein By way of example, a screening method will comprise isolating the coding region of the cell cycle protein gene using the known DNA sequence to synthesize a selected probe of about 40 bases Hybridization probes may be labeled by a variety of labels, including radionucleotides such as ³ P or ³⁵S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems Labeled probes having a sequence complementary to that of the cell cycle protein gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes

Nucleotide sequences encoding a cell cycle protein can also be used to construct hybridization probes for mapping the gene which encodes that cell cycle protein and for the genetic analysis of individuals with genetic disorders The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries Nucleic acids which encode cell cycle protein or its modified forms can also be used to generate either transgenic animals or knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents A transgenic animal (e g , a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animai or an ancestor of the animal at a prenatal, e g , an embryonic stage A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops In one embodiment, cDNA encoding a cell cycle protein can be used to clone genomic DNA encoding a cell cycle protein in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express the desired DNA Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U S Patent Nos 4,736,866 and 4,870,009 Typically, particular cells would be targeted for the cell cycle protein transgene incorporation with tissue-specific enhancers Transgenic animals that include a copy of a transgene encoding a cell cycle protein introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of the desired nucleic acid Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition

Alternatively, non-human homologues of the cell cycle protein can be used to construct a cell cycle protein "knock out" animal which has a defective or altered gene encoding a cell cycle protein as a result of homologous recombination between the endogenous gene encoding a ceil cycle protein and altered genomic DNA encoding a cell cycle protein introduced into an embryonic cell of the animal For example, cDNA encoding a cell cycle protein can be used to clone genomic DNA encoding a cell cycle protein in accordance with established techniques A portion of the genomic DNA encoding a cell cycle protein can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e g , Thomas and Capecchi, Cell, 51 503 (1987) for a description of homologous recombination vectors] The vector is introduced into an embryonic stem cell line (e g , by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e g , Li et al , Cell, 69 915 (1992)] The selected cells are then injected into a blastocyst of an animal (e g , a mouse or rat) to form aggregation chimeras [see e g , Bradley, in Teratocarcmomas and Embryonic Stem Cells A Practical Approach, E J Robertson, ed (IRL, Oxford, 1987), pp 113-152] A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the cell cycle protein

It is understood that the models described herein can be varied For example, "knock-in" models can be formed, or the models can be cell-based rather than animal models

Nucleic acid encoding the cell cycle polypeptides, antagonists or agonists may also be used in gene therapy In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane (Zamecnik et al , Proc Natl Acad Sci USA 83, 4143-4146 [1986]) The oligonucleotides can be modified to enhance their uptake, e g by substituting their negatively charged phosphodiester groups by uncharged groups

There are a variety of techniques available for introducing nucleic acids into viable cells The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al , Trends in Biotechnology 1 1 , 205-210 [1993]) In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e g capsid proteins or fragments thereof tropic for a particular ceil type, antibodies for proteins which undergo mternalization in cycling, proteins that target intracellular localization and enhance intracellular half-life The technique of receptor-mediated endocytosis is described, for example, by Wu e: a/ , J_ Bid. Chem 262, 4429-4432 (1987), and Wagner et al , Proc Natl Acad Sci USA 87, 3410-3414 (1990) For review of gene marking and gene therapy protocols see Anderson et al , Science 256 808-813 (1992)

In a preferred embodiment, the cell cycle proteins, nucleic acids, variants, modified proteins, cells and/or transgenics containing the said nucleic acids or proteins are used in screening assays Identification of the cell cycle protein provided herein permits the design of drug screening assays for compounds that bind or interfere with binding to the cell cycle protein, modulate cell cycle protein activity, and modulate cell cycle activity

The assays described herein preferably utilize the human cell cycle protein, although other mammalian proteins may also be used, including rodents (mice, rats, hamsters, guinea pigs, etc ), farm animals (cows, sheep, pigs, horses, etc ) and primates These latter embodiments may be preferred in the development of animal models of human disease In some embodiments, as outlined herein, variant or derivative cell cycle proteins may be used, including deletion cell cycle proteins as outlined above

In a preferred embodiment, the methods comprise combining a cell cyle protein and a candidate bioactive agent, and determining the binding of the candidate agent to the cell cycle protein In other embodiments, further discussed below, binding interference or bioactivity is determined

The term candidate bioactive agent or "exogeneous compound" as used herein describes any molecule, e g , protein, small organic molecule, carbohydrates (including polysacchaπdes), pdynucleotide, lipids, etc Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations Typically, one of these concentrations serves as a negative control, / e , at zero concentration or below the level of detection In addition, positive controls, i e the use of agents known to alter cell cycling, may be used For example, p21 is a molecule known to arrest cells in the G1 cell phase, by binding G1 cycl -CDK complexes

Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amme, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups Candidate agents are also found among biomolecules including peptides, sacchaπdes, fatty acids, steroids, puπnes, pyπmidines, derivatives, structural analogs or combinations thereof Particularly preferred are peptides

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esteπfication, amidification to produce structural analogs

In a preferred embodiment, a library of different candidate bioactive agents are used Preferably, the library should provide a sufficiently structurally diverse population of randomized agents to effect a probabilistically sufficient range of diversity to allow binding to a particular target Accordingly, an interaction library should be large enough so that at least one of its members will have a structure that gives it affinity for the target Although it is difficult to gauge the required absolute size of an interaction library, nature provides a hint with the immune response a diversity of 10⁷-10⁸ different antibodies provides at least one combination with sufficient affinity to interact with most potential antigens faced by an organism Published in vitro selection techniques have also shown that a library size of 10⁷ to 10⁸ is sufficient to find structures with affinity for the target A library of all combinations of a peptide 7 to 20 ammo acids in length, such as generally proposed herein, has the potential to code for 20⁷ (10⁹) to 20²⁰ Thus, with libraries of 10⁷ to 10⁸ different molecules the present methods allow a 'working" subset of a theoretically complete interaction library for 7 amino acids, and a subset of shapes for the 20²⁰ library Thus, in a preferred embodiment, at least 10⁶, preferably at least 10⁷, more preferably at least 10⁸ and most preferably at least 10⁹ different sequences are simultaneously analyzed in the subject methods Preferred methods maximize library size and diversity

In a preferred embodiment, the candidate bioactive agents are proteins By "protein" herein is meant at least two covalently attached ammo acids, which includes proteins, polypeptides, oligopeptides and peptides The protein may be made up of naturally occurring am o acids and peptide bonds or synthetic peptidomimetic structures Thus "ammo acid", or "peptide residue", as used herein means both naturally occurring and synthetic ammo acids For example, homo- phenylalanme, citrulline and noreleucine are considered ammo acids for the purposes of the invention "Ammo acid" also includes imino acid residues such as proline and hydroxyprolme The side chains may be in either the (R) or the (S) configuration In the preferred embodiment, the ammo acids are in the (S) or L-configuration If non-naturally occurring side chains are used, non- am o acid substituents may be used, for example to prevent or retard in vivo degradations Chemical blocking groups or other chemical substituents may also be added

In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used In this way libraries of procaryotic and eukaryotic proteins may be made for screening in the systems described herein Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred In a preferred embodiment, the candidate bioactive agents are peptides of from about 5 to about 30 am o acids, with from about 5 to about 20 ammo acids being preferred, and from about 7 to about 15 being particularly preferred The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and ammo acids, respectively Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or ammo acid at any position The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents

In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position In a preferred embodiment, the library is biased That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities For example, in a preferred embodiment, the nucleotides or am o acid residues are randomized within a defined class, for example, of hydrophobic ammo acids, hydrophilic residues, steπcally biased (either small or large) residues, towards the creation of cyste es, for cross-linking, pralines for SH- 3 domains, seπnes, threonines, tyrosines or histid es for phosphorylation sites, etc , or to puπnes, etc

In a preferred embodiment, the candidate bioactive agents are nucleic acids By "nucleic acid" or "oligonucleotide" or grammatical equivalents herein means at least two nucleotides covalently linked together A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage, et al , Tetrahedron, 49(10) 1925 (1993) and references therein, Letsmger, J Org Chem , 35 3800 (1970), Spπnzl, et al , Eur J Biochem , 81 579 (1977), Letsmger, et al . Nucl Acids Res , 14 3487 (1986), Sawai, et al , Chem Lett , 805 (1984), Letsmger, et al . J Am Chem Soc , 1104470 (1988), and Pauwels, et al , Chemica Scnpta, 26 141 (1986)), phosphorothioate (Mag, et al , Nucleic Acids Res , 19 1437 (1991 ), and U S Patent No 5,644,048), phosphorodithioate (Bπu, et al , J Am Chem Soc , 111 2321 (1989)), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J_ Am Chem Soc , 114 1895 (1992), Meier, et al , Chem Int Ed Enql , 31 1008 (1992), Nielsen, Nature, 365 566 (1993), Carlsson et al , Nature, 380 207 (1996), all of which are incorporated by reference)) Other analog nucleic acids include those with positive backbones (Denpcy, ef al , Proc Natl Acad Sci USA 92 6097 ( 1995)), non- ionic backbones (U S Patent Nos 5,386,023, 5,637,684, 5,602,240, 5,216,141 , and 4,469,863, Kiedrowshi, et al , Anqew Chem Intl Ed English, 30 423 (1991 ), Letsmger, et al , ___ Am. Chem Soc . 110 4470 (1988), Letsmger, et al , Nucleoside & Nucleotide, 13 1597 (1994), Chapters 2 and 3, ASC Symposium Series 580 'Carbohydrate Modifications in Antisense Research", Ed Y S Sanghui and P Dan Cook, Mesmaeker, et al , Bioorganic & Medicinal Chem Lett., 4.395 (1994); Jeffs, et al., J Biomolecular NMR, 34 17 (1994), Tetrahedron Lett , 37 743 (1996)) and non-πbose backbones, including those described in U S Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y S Sanghui and P Dan Cook Nucleic acids containing one or more carbocyc c sugars are also included within the definition of nucleic acids (see Jenkins, et al , Chem Soc Rev , (1995) pp. 169- 176) Several nucleic acid analogs are described in Rawls, C & E News, June 2, 1997, page 35 All of these references are hereby expressly incorporated by reference These modifications of the πbose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments. In addition, mixtures of naturally occurring nucleic acids and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyπbo- and πbo-nucleotides, and any combination of bases, including uracil, adenine, thymme, cytosme, guanme, mosine, xathan e hypoxathanine, isocytosme, isoguanme, etc.

As described above generally for proteins, nucleic acid candidate bioactive agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of procaryotic or eukaryotic genomes may be used as is outlined above for proteins.

In a preferred embodiment, the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature

In a preferred embodiment, the candidate bioactive agents are linked to a fusion partner By "fusion partner" or "functional group" herein is meant a sequence that is associated with the candidate bioactive agent, that confers upon all members of the library in that class a common function or ability Fusion partners can be heterologous (i e. not native to the host cell), or synthetic (not native to any cell) Suitable fusion partners include, but are not limited to: a) presentation structures, which provide the candidate bioactive agents in a conformationally restricted or stable form, b) targeting sequences, which allow the localization of the candidate bioactive agent into a subcellular or extracellular compartment, c) rescue sequences which allow the purification or isolation of either the candidate bioactive agents or the nucleic acids encoding them, d) stability sequences, which confer stability or protection from degradation to the candidate bioactive agent or the nucleic acid encoding it, for example resistance to proteolytic degradation, e) dimerization sequences, to allow for peptide dimerization, or f) any combination of a), b), c), d), and e), as well as linker sequences as needed In one embodiment of the methods described herein, portions of cell cycle proteins are utilized, in a preferred embodiment, portions having cell cycle activity are used Cell cycle activity is described further below and includes binding activity to at least one lAPs or cell cycle protein modulators as further described below In addition, the assays described herein may utilize either isolated cell cycle proteins or cells comprising the cell cycle proteins

Generally, in a preferred embodiment of the methods herein, for example for binding assays, the cell cycle protein or the candidate agent is non-diffusibly bound to an insoluble support having isolated sample receiving areas (e g a microtiter plate, an array, etc ) The insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening The surface of such supports may be solid or porous and of any convenient shape Examples of suitable insoluble supports include microtiter plates arrays, membranes and beads These are typically made of glass, plastic (e g , polystyrene), polysacchaπdes, nylon or nitrocellulose, teflon™, etc Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples In some cases magnetic beads and the like are included The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable Preferred methods of binding include the use of antibodies (which do not steπcally block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslmking, the synthesis of the protein or agent on the surface, etc In some embodiments, lAPs can be used Following binding of the protein or agent, excess unbound material is removed by washing The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety Also included in this invention are screening assays wherein solid supports are not used, examples of such are described below

In a preferred embodiment, the cell cycle protein is bound to the support, and a candidate bioactive agent is added to the assay Alternatively, the candidate agent is bound to the support and the cell cycle protein is added Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc Of particular interest are screening assays for agents that have a low toxicity for human cells A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc ) and the like

The determination of the binding of the candidate bioactive agent to the celi cycle protein may be done in a number of ways In a preferred embodiment, the candidate bioactive agent is labelled, and binding determined directly For example this may be done by attaching all or a portion of the cell cycle protein to a solid support, adding a labelled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support Various blocking and washing steps may be utilized as is known in the art

By "labeled" herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e g radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc Specific binding molecules include pairs, such as biotm and streptavidm, digoxin and antidigoxin etc For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above The label can directly or indirectly provide a detectable signal

In some embodiments, only one of the components is labeled For example, the proteins (or proteinaceous candidate agents) may be labeled at tyrosine positions using ¹²⁵l, or with fluorophores Alternatively, more than one component may be labeled with different labels, using ¹²⁵l for the proteins, for example and a fluorophor for the candidate agents

In a preferred embodiment, the binding of the candidate bioactive agent is determined through the use of competitive binding assays In this embodiment, the competitor is a binding moiety known to bind to the target molecule (i e cell cycle protein), such as an antibody, peptide, binding partner, ligand, etc In a preferred embodiment, the competitor is lAPs Under certain circumstances, there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent This assay can be used to determine candidate agents which interfere with binding between cell cycle proteins and lAPs "Interference of binding" as used herein means that native binding of the cell cycle protein differs in the presence of the candidate agent The binding can be eliminated or can be with a reduced affinity Therefore, in one embodiment, interference is caused by, for example, a conformation change, rather than direct competition for the native binding site

In one embodiment, the candidate bioactive agent is labeled Either the candidate bioactive agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40°C Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening Typically between 0 1 and 1 hour will be sufficient Excess reagent is generally removed or washed away The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding

In a preferred embodiment, the competitor is added first, followed by the candidate bioactive agent Displacement of the competitor is an indication that the candidate bioactive agent is binding to the cell cycle protein and thus is capable of binding to, and potentially modulating, the activity of the cell cycle protein In this embodiment, either component can be labeled. Thus, for example, if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent Alternatively, if the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement

in an alternative embodiment, the candidate bioactive agent is added first, with incubation and washing, followed by the competitor The absence of binding by the competitor may indicate that the bioactive agent is bound to the cell cycle protein with a higher affinity Thus, if the candidate bioactive agent is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the candidate agent is capable of binding to the cell cycle protein

In a preferred embodiment, the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the cell cycle proteins Such assays can be done with the cell cycle protein or cells compπsing said cell cycle protein In one embodiment, the methods comprise combining an cell cycle protein and a competitor in a first sample A second sample comprises a candidate bioactive agent, an cell cycle protein and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the cell cycle protein and potentially modulating its activity That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the cell cycle protein.

Alternatively, a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native cell cycle protein, but cannot bind to modified cell cycle proteins The structure of the cell cycle protein may be modeled, and used in rational drug design to synthesize agents that interact with that site Drug candidates that affect cell cycle bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein

Positive controls and negative controls may be used in the assays Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound

A variety of other reagents may be included in the screening assays These include reagents like salts, neutral proteins, e g albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc , may be used The mixture of components may be added in any order that provides for the requisite binding

Screening for agents that modulate the activity of a cell cycle protein may also be done In a preferred embodiment methods for screening for a bioactive agent capable of modulating the activity of a cell cycle protein comprise the steps of adding a candidate bioactive agent to a sample of a cell cycle protein (or cells comprising a cell cycle protein) and determining an alteration in the biological activity of the cell cycle protein "Modulating the activity of a cell cycle protein" includes an increase in activity, a decrease in activity or a change in the type or kind of activity present Thus, in this embodiment, the candidate agent may bind to a cell cycle protein (although this may not be necessary), and should alter its biological or biochemical activity as defined herein The methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of cell cycle protein

Thus, in this embodiment, the methods comprise combining an cell cycle protein sample and a candidate bioactive agent, and evaluating the effect on the activity of the cell cycle protein By "cell cycle protein activity" or grammatical equivalents herein is meant at least one of the cell cycle protein's biological activities, including, but not limited to, its ability to affect the cell cycle, bind to lAPs, suppress tumor growth, stimulate or enhance cell proliferation, activate p53 binding site controlled promoters, modulate apoptosis, and/or modulate cellular responses to stress

In a preferred embodiment, the activity of the cell cycle protein is decreased, in another preferred embodiment, the activity of the cell cycle protein is increased Thus, bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments

In a preferred embodiment, the invention provides methods for screening for bioactive agents capable of modulating the activity of an cell cycle protein The methods comprise adding a candidate bioactive agent, as defined above to a cell comprising cell cycle proteins Preferred cell types include almost any cell The cells contain a recombinant nucleic acid that encodes an cell cycle protein In a preferred embodiment, a library of candidate agents are tested on a plurality of cells

In a preferred embodiment, the present invention provides a cell cycle protein and a cell cycle nucleic acid encoding a cell cycle protein which proteins modulate apoptosis in a eukaryotic cell, more preferably a mammalian cell This modulation of apoptosis preferably involves interaction between a cell cycle protein and an IAP, and more preferably involves modulation of the ubiquitination state of an IAP by a cell cycle protein In one embodiment, this modulation of ubiquitination occurs through modulation of ubiquitin protein ligase activity or ubiquitin conjugating activity of an IAP (activities described by Yang et al Science 288 874-877, 2000, and Hauser et al JCB 141 1415-1422, 2000, both incorporated herein in their entirety by reference) by a cell cycle protein In a preferred embodiment, modulation of the ubiquitination state of an IAP by a cell cycle protein affects degradation of the IAP

In a preferred embodiment, modulation of apoptosis by a cell cycle protein involves the modulation of caspase activity The modulation of caspase activity by a cell cycle protein preferably occurs through modulation of the ubiquitination state of a caspase by a cell cycle protein, preferably through modulation of ubiquitin protein ligase activity or ubiquitin conjugating activity of an IAP in respect of a caspase substrate by a cell cycle protein, and more preferably where such modulation of the ubiquitination state of a caspase affects its degradation

In one embodiment, the present invention provides a cell cycle protein and a cell cycle nucleic acid encoding a cell cycle protein which proteins modulate apoptosis, which modulation involves the recruitment of an IAP to p53 or a p53-contaιnιng protein conglomerate By protein conglomerate is meant an assembly of proteins which proteins interact molecularly with at least one other member of the assembly which assembly forms a functional or multifunctional unit, not entirely unlike assemblies of protein subunits which form hdoenzymes In a preferred embodiment, the recruited IAP modulates ubiquitination of a component of a p53-contaιnιng protein conglomerate In a preferred embodiment, the recruited IAP modulates ubiquitination of p53 In one embodiment, a cell cycle protein modulates ubiquitination of p53 in response to an extrinsic cue, for example as in the response of thymocytes to an extrinsic cue which response is characterized by apoptosis In one embodiment, a cell cycle protein modulates ubiquitination of p53 in response to an intrinsic cue, for example as in response to activation of the ras signal transduction pathway and changes in the phosphorylation state of intracellular molecules In a preferred embodiment, the modulation of p53 ubiquitination by a cell cycle protein affects p53 degradation in a preferred embodiment, modulation of apoptosis by a cell cycle protein involves cell cycle protein modulation of p53 degradation ana expression of p53 target genes In a preferred embodiment p53 target genes include the cdk inhibitor p27 and members of the Bcl2 gene family

In one embodiment, the present invention provides a cell cycle protein and a cell cycle nucleic acid encoding a cell cycle protein which proteins modulate apoptosis by recruiting a caspase to the nucleus, preferably via association with an IAP In a preferred embodiment, the recruited caspase effects at least one proteolytic reaction in the nucleus which reaction is characteristic of apoptosis

In one embodiment, the present invention provides a cell cycle protein and a cell cycle nucleic acid encoding a cell cycle protein which proteins modulate progression through the ceil cycle, which modulation involves the recruitment of an IAP to p53 or a p53-contaιnιng protein conglomerate In a preferred embodiment, the recruited IAP modulates ubiquitination of a component of a p53- containing protein conglomerate In a preferred embodiment, the recruited IAP modulates ubiquitination of p53 In one embodiment, a cell cycle protein modulates ubiquitination of p53 in response to an extrinsic cue, for example in response to binding of an extracellular ligand to a receptor at the cell surface In one embodiment, a cell cycle protein modulates ubiquitination of p53 in response to an intrinsic cue for example as in response to activation of the JAK/STAT signal transduction pathway and changes in the phosphorylation state of intracellular molecules In a preferred embodiment, the modulation of p53 ubiquitination by a cell cycle protein affects p53 degradation In a preferred embodiment, modulation of progression through the cell cycle by a cell cycle protein involves cell cycle protein modulation of p53 degradation and expression of p53 target genes In a preferred embodiment p53 target genes include the cdk inhibitor p27 and members of the Bcl2 gene family

Screening for agents that modulate progression through the cell cycle, or modulate cell cycle activity, may also be done Detection of cell cycle regulation may be done as will be appreciated by those in the art In one embodiment, indicators of the cell cycle are used There are a number of parameters that may be evaluated or assayed to allow the detection of alterations in cell cycle regulation, including, but not limited to, cell viability assays, assays to determine whether cells are arrested at a particular cell cycle stage ( 'cell proliferation assays '), and assays to determine at which cell stage the cells have arrested ("cell phase assays") By assaying or measuring one or more of these parameters, it is possible to detect not only alterations in cell cycle regulation, but alterations of different steps of the cell cycle regulation pathway This may be done to evaluate native cells, for example to quantify the aggressiveness of a tumor cell type, or to evaluate the effect of candidate drug agents that are being tested for their effect on cell cycle regulation In this manner, rapid, accurate screening of candidate agents may be performed to identify agents that modulate cell cycle regulation

Thus, the present compositions and methods are useful to elucidate bioactive agents that can cause a cell or a population of cells to either move out of one growth phase and into another, or arrest in a growth phase In some embodiments, the cells are arrested in a particular growth phase, and it is desirable to either get them out of that phase or into a new phase Alternatively, it may be desirable to force a cell to arrest in a phase, for example G1 , rather than continue to move through the cell cycle Similarly, it may be desirable in some circumstances to accelerate a non- arrested but slowly moving population of cells into either the next phase or just through the cell cycle, or to delay the onset of the next phase For example it may be possible to alter the activities of certain enzymes, for example kinases, phosphatases, proteases or ubiquitination enzymes, that contribute to initiating cell phase changes

In a preferred embodiment, the methods outlined herein are done on cells that are not arrested in the G1 phase, that is, they are rapidly or uncontrollably growing and replicating, such as tumor cells In this manner, candidate agents are evaluated to find agents that can alter the cell cycle regulation, i e cause the cells to arrest at cell cycle checkpoints, such as in G1 (although arresting in other phases such as S, G2 or M are also desirable) Alternatively, candidate agents are evaluated to find agents that can cause proliferation of a population of cells, i e that allow cells that are generally arrested in G1 to start proliferating again, for example, peripheral blood cells, terminally differentiated cells, stem cells in culture, etc

Accordingly, the invention provides methods for screening for alterations in cell cycle regulation of a population of cells By "alteration" or "modulation" (used herein interchangeably), is generally meant one of two things In a preferred embodiment, the alteration results in a change in the cell cycle of a ceil, i e a proliferating cell arrests in any one of the phases, or an arrested cell moves out of its arrested phase and starts the cell cycle, as compared to another cell or in the same cell under different conditions Alternatively, the progress of a cell through any particular phase may be altered, that is, there may be an acceleration or delay in the length of time it takes for the cells to move thorough a particular growth phase For example, the cell may be normally undergo a G1 phase of several hours, the addition of an agent may prolong the G1 phase

The measurements can be determined wherein all of the conditions are the same for each measurement, or under various conditions, with or without bioactive agents, or at different stages of the cell cycle process For example, a measurement of cell cycle regulation can be determined in a ceil or cell population wherem a candidate bioactive agent is present and wherein the candidate bioactive agent is absent In another example, the measurements of cell cycle regulation are determined wherein the condition or environment of the cell or populations of cells differ from one another For example, the cells may be evaluated in the presence or absence or previous or subsequent exposure of physiological signals, for example hormones, antibodies, peptides, antigens, cytokmes, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcmogenics, or other cells (i e cell-cell contacts) In another example, the measurements of cell cycle regulation are determined at different stages of the cell cycle process In yet another example, the measurements of cell cycle regulation are taken wherein the conditions are the same, and the alterations are between one cell or cell population and another cell or cell population

By a ' population of cells" or "library of cells" herein is meant at least two cells, with at least about 10³ being preferred, at least about 10⁶ being particularly preferred, and at least about 10⁸ to 10⁹ being especially preferred The population or sample can contain a mixture of different cell types from either primary or secondary cultures although samples containing only a single cell type are preferred, for example, the sample can be from a cell line, particularly tumor cell lines, as outlined below The cells may be in any cell phase, either synchronously or not, including M, G1 , S, and G2 In a preferred embodiment, cells that are replicating or proliferating are used, this may allow the use of retroviral vectors for the introduction of candidate bioactive agents Alternatively, non- replicating cells may be used, and other vectors (such as adenovirus and Ientivirus vectors) can be used In addition, although not required, the cells are compatible with dyes and antibodies Preferred cell types for use in the invention mcluαe, but are not limited to, mammalian cells, including animal (rodents, including mice, rats, hamsters and gerbils), primates, and human cells, particularly including tumor cells of all types, including breast, skin, lung, cervix, colonrectal, leukemia, brain, etc

In a preferred embodiment, the methods comprise assaying one or more of several different cell parameters, including, but not limited to, cell viability, cell proliferation, and cell phase Other parameters include but not limited to, its ability to affect the cell cycle, binding to at least one lAPs, suppression of tumor growth, activation of p53 binding site controlled promoters, modulation of apoptosis, and/or modulation of cellular responses to stress

In a preferred embodiment, cell viability is assayed, to ensure that a lack of cellular change is due to experimental conditions (i e the introduction of a candidate bioactive agent) not cell death There are a variety of suitable cell viability assays which can be used, including, but not limited to, light scattering, viability dye staining, and exclusion dye staining

In a preferred embodiment, a light scattering assay is used as the viability assay, as is well known in the art For example, when viewed in the FACS, cells have particular characteristics as measured by their forward and 90 degree (side) light scatter properties These scatter properties represent the size, shape and granule content of the cells These properties account for two parameters to be measured as a readout for the viability Briefly, the DNA of dying or dead cells generally condenses, which alters the 90^° scatter, similarly, membrane blebb g can alter the forward scatter Alterations in the intensity of light scattering, or the cell-refractive index indicate alterations in viability

Thus, in general, for light scattering assays, a live cell population of a particular cell type is evaluated to determine it's forward and side scattering properties This sets a standard for scattering that can subsequently be used

In a preferred embodiment, the viability assay utilizes a viability dye There are a number of known viability dyes that stain dead or dying cells, but do not stain growing cells For example, annexin V is a member of a protein family which displays specific binding to phospholipid (phosphotidylseπne) in a divalent ion dependent manner This protein has been widely used for the measurement of apoptosis (programmed cell death) as cell surface exposure of phosphatidylseπne is a hallmark early signal of this process Suitable viability dyes include, but are not limited to, annexin, ethidium homoαιmer-1 , DEAD Red, propidium iodide, SYTOX Green, etc , and others known in the art, see the Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Haugland, Sixth Edition, hereby incorporated by reference, see Apoptosis Assay on page 285 in particular, and Chapter 16 Protocols for viability dye staining for cell viability are known see Molecular Probes catalog supra In this embodiment, the viability dye such as annexin is labeled, either directly or indirectly, and combined with a cell population Annexin is commercially available, i e , from PharMmgen, San Diego, California, or Caltag Laboratories, Millbrae, California Preferably, the viability dye is provided in a solution wherein the dye is in a concentration of about 100 ng/ml to about 500 ng/ml, more preferably, about 500 ng/ml to about 1 μg/ml, and most preferably, from about 1 μg/ml to about 5 μg/ml In a preferred embodiment, the viability dye is directly labeled, for example annexin may be labeled with a fluorochrome such as fluorecem isothiocyanate (FITC), Alexa dyes, TRITC, AMCA, APC, tπ-color, Cy-5, and others known in the art or commercially available In an alternate preferred embodiment, the viability dye is labeled with a first label, such as a hapten such as biotin, and a secondary fluorescent label is used, such as fluorescent streptavidin Other first and second labeling pairs can be used as will be appreciated by those in the art

Once added, the viability dye is allowed to incubate with the cells for a period of time, and washed, if necessary The cells are then sorted as outlined below to remove the non-viable cells

In a preferred embodiment, exclusion dye staining is used as the viability assay Exclusion dyes are those which are excluded from living ceils, i e they are not taken up passively (they do not permeate the cell membrane of a live cell) However, due to the permeability of dead or dying cells, they are taken up by dead cells Generally, but not always, the exclusion dyes bind to DNA, for example via intercalation Preferably, the exclusion dye does not fluoresce, or fluoresces poorly, in the absence of DNA, this eliminates the need for a wash step Alternatively, exclusion dyes that require the use of a secondary label may also be used Preferred exclusion dyes include, but are not limited to, ethidium bromide, ethidium homodιmer-1 , propidium iodine, SYTOX green nucleic acid stain, Calcem AM, BCECF AM, fluorescein diacetate, TOTO® and TO-PRO™ (from Molecular Probes, supra, see chapter 16) and others known in the art

Protocols for exclusion dye staining for cell viability are known, see the Molecular Probes catalog, supra In general, the exclusion dye is added to the cells at a concentration of from about 100 ng/ml to about 500 ng/ml, more preferably, about 500 ng/ml to about 1 μg/ml, and most preferably from about 0 1 μg/ml to about 5 μg/mi, with about 0 5 μg/ml being particularly preferred The cells and the exclusion dye are incubated for some period of time, washed, if necessary, and then the cells sorted as outlined below, to remove non-viable cells from the population

In addition, there are other cell viability assays which may be run, including for example enzymatic assays, which can measure extracellular enzymatic activity of either live cells (i e secreted proteases, etc ) or dead cells (i e the presence of intracellular enzymes in the media, for example intracellular proteases, mitochondria! enzymes, etc ) See the Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Haugland, Sixth Edition, hereby incorporated by reference, see chapter 16 in particular In a preferred embodiment, at least one cell viability assay is run, with at least two different cell viability assays being preferred, when the fluors are compatible When only 1 viability assay is run, a preferred embodiment utilizes light scattering assays (both forward and side scattering) When two viability assays are run, preferred embodiments utilize light scattering and dye exclusion, with light scattering and viability dye staining also possible, and all three being done in some cases as well Viability assays thus allow the separation of viable cells from non-viable or dying cells

In addition to a cell viability assay, a preferred embodiment utilizes a cell proliferation assay. By "proliferation assay" herein is meant an assay that allows the determination that a cell population is either proliferating, i e replicating, or not replicating

In a preferred embodiment, the proliferation assay is a dye inclusion assay A dye inclusion assay relies on dilution effects to distinguish between cell phases Briefly, a dye (generally a fluorescent dye as outlined below) is introduced to cells and taken up by the cells Once taken up, the dye is trapped in the cell, and does not diffuse out As the cell population divides, the dye is proportionally diluted That is, after the introduction of the inclusion dye, the cells are allowed to incubate for some period of time, cells that lose fluorescence over time are dividing, and the cells that remain fluorescent are arrested in a non-growth phase

Generally, the introduction of the inclusion dye may be done in one of two ways Either the dye cannot passively enter the ceils (e g it is charged), and the cells must be treated to take up the dye; for example through the use of a electric pulse Alternatively, the dye can passively enter the cells, but once taken up, it is modified such that it cannot diffuse out of the cells For example, enzymatic modification of the inclusion dye may render it charged, and thus unable to diffuse out of the cells For example, the Molecular Probes CellTracker™ dyes are fluorescent chloromethyl derivatives that freely diffuse into cells, and then glutathione S-transferase-mediated reaction produces membrane impermeant dyes

Suitable inclusion dyes include, but are not limited to, the Molecular Probes line of CellTracker™ dyes , including, but not limited to CellTracker™ Blue, CellTracker™ Yellow-Green, CellTracker™ Green, CellTracker™ Orange, PKH26 (Sigma), and others known in the art; see the Molecular Probes Handbook, supra; chapter 15 in particular

In general, inclusion dyes are provided to the cells at a concentration ranging from about 100 ng/ml to about 5 μg/ml, with from about 500 ng/ml to about 1 μg/mi being preferred A wash step may or may not be used In a preferred embodiment, a candidate bioactive agent is combined with the cells as descπbed herein The cells and the inclusion dye are incubated for some period of time, to allow cell division and thus dye dilution The length of time will depend on the cell cycle time for the particular cells, in general, at least about 2 cell divisions are preferred, with at least about 3 being particularly preferred and at least about 4 being especially preferred The cells are then sorted as outlined below, to create populations of cells that are replicating and those that are not. As will be appreciated by those in the art, in some cases, for example when screening for anti- proliferation agents, the bright (i e fluorescent) cells are collected, in other embodiments, for example for screening for proliferation agents the low fluorescence cells are collected Alterations are determined by measuring the fluorescence at either different time points or in different cell populations, and comparing the determinations to one another or to standards

In a preferred embodiment, the proliferation assay is an antimetaboiite assay In general, antimetaboiite assays find the most use when agents that cause cellular arrest in G1 or G2 resting phase is desired In an antimetaboiite proliferation assay, the use of a toxic antimetaboiite that will kill dividing cells will result in survival of only those ceils that are not dividing Suitable antimetabolites include, but are not limited to, standard chemotherapeutic agents such as methotrexate, cisplatm, taxol, hydroxyurea, nucleotide analogs such as AraC, etc In addition, antimetaboiite assays may include the use of genes that cause cell death upon expression

The concentration at which the antimetaboiite is added will depend on the toxicity of the particular antimetaboiite, and will be determined as is known in the art The antimetaboiite is added and the cells are generally incubated for some peπod of time, again, the exact period of time will depend on the characteristics and identity of the antimetaboiite as well as the cell cycle time of the particular cell population Generally, a time sufficient for at least one cell division to occur

In a preferred embodiment, at least one proliferation assay is run, with more than one being preferred Thus, a proliferation assay results in a population of proliferating cells and a population of arrested cells Moreover, other proliferation assays may be used, i e , coloπmetπc assays known in the art

In a preferred embodiment either after or simultaneously with one or more of the proliferation assays outlined above, at least one cell phase assay is done A "cell phase" assay determines at which cell phase the cells are arrested, M, G1 , S, or G2

In a preferred embodiment, the cell phase assay is a DNA binding dye assay Briefly, a DNA binding dye is introduced to the cells, and taken up passively Once inside the cell the DNA binding dye binds to DNA, generally by intercalation, although in some cases, the dyes can be either major or minor groove binding compounds The amount of dye is thus directly correlated to the amount of DNA in the cell, which varies by cell phase, G2 and M phase cells have twice the DNA content of G1 phase cells, and S phase cells have an intermediate amount, depending on at what point in S phase the cells are Suitable DNA binding dyes are permeant, and include, but are not limited to, Hoechst 33342 and 33258, acπdine orange, 7-AAD, LDS 751 , DAPI, and SYTO 16, Molecular Probes Handbook, supra, chapters 8 and 16 in particular In general, the DNA binding dyes are added in concentrations ranging from about 1 μg/ml to about 5 μg/ml The dyes are added to the cells and allowed to incubate for some period of time, the length of time will depend in part on the dye chosen In one embodiment, measurements are taken

immediately after addition of the dye The cells are then sorted as outlined below, to create populations of cells that contain different amounts of dye, and thus different amounts of DNA, in this way, cells that are replicating are separated from those that are not As will be appreciated by those in the art, in some cases, for example wnen screening for anti-proliferation agents, cells with the least fluorescence (and thus a single copy of the genome) can be separated from those that are replicating and thus contain more than a single genome of DNA Alterations are determined by measuring the fluorescence at either different time points or in different cell populations, and comparing the determinations to one another or to standards

In a preferred embodiment, the cell phase assay is a cyclin destruction assay In this embodiment, prior to screening (and generally prior to the introduction of a candidate bioactive agent, as outlined below), a fusion nucleic acid is introduced to the cells The fusion nucleic acid comprises nucleic acid encoding a cyclin destruction box and a nucleic acid encoding a detectable molecule "Cyclin destruction boxes" are known in the art and are sequences that cause destruction via the ubiquitination pathway of proteins containing the boxes during particular cell phases That is, for example, G1 cyclms may be stable during G1 phase but degraded during S phase due to the presence of a G1 cyclin destruction box Thus, by linking a cyclin destruction box to a detectable molecule, for example green fluorescent protein, the presence or absence of the detectable molecule can serve to identify the cell phase of the cell population In a preferred embodiment, multiple boxes are used, preferably each with a different fluor, such that detection of the cell phase can occur

A number of cyclin destruction boxes are known in the art, for example, cyclin A has a destruction box comprising the sequence RTVLGVIGD, the destruction box of cyclin B1 comprises the sequence RTALGDIGN See Glotzer et al , Nature 349 132-138 (1991 ) Other destruction boxes are known as well YMTVSIIDRFMQDSCVPKKMLQLVGVT (rat cyclin B),

KFRLLQETMYMTVSIIDRFMQNSCVPKK (mouse cyclin B),

RAILIDWLIQVQMKFRLLQETMYMTVS (mouse cyclin B1 ), DRFLQAQLVCRKKLQVVGITALLLASK (mouse cyclin B2), and MSVLRGKLQLVGTAAMLL (mouse cyclin A2)

The nucleic acid encoding the cyclin destruction box is operably linked to nucleic acid encoding a detectable molecule The fusion proteins are constructed by methods known in the art For example, the nucleic acids encoding the destruction box is ligated to a nucleic acid encoding a detectable molecule By "detectable molecule herein is meant a molecule that allows a cell or compound comprising the detectable molecule to be distinguished from one that does not contain it, i e , an epitope, sometimes called an antigen TAG, a specific enzyme, or a fluorescent molecule Preferred fluorescent molecules include but are not limited to green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP) red fluorescent protein (RFP), and enzymes including luciferase and β-galactosidase When antigen TAGs are used, preferred embodiments utilize cell surface antigens The epitope is preferably any detectable peptide which is not generally found on the cytoplasmic membrane, although in some instances, if the epitope is one normally found on the cells, increases may be detected, although this is generally not preferred Similarly, enzymatic detectable molecules may also be used, for example, an enzyme that generates a novel or chromogenic product

Accordingly, the results of sorting after cell phase assays generally result in at least two populations of cells that are in different cell phases

The proteins and nucleic acids provided herein can also be used for screening purposes wherein the protein-protein interactions of the cell cycle proteins can be identified Genetic systems have been described to detect protein-protein interactions The first work was done in yeast systems, namely the "yeast two-hybrid" system The basic system requires a protein-protein interaction in order to turn on transcription of a reporter gene Subsequent work was done in mammalian cells See Fields et al Nature 340 245 (1989), Vasavada et al , PNAS USA 88 10686 (1991 ), Fearon et al , PNAS USA 89 7958 (1992), Dang et al , Mol Cell Bid 1 1 954 (1991 ), Chien et al , PNAS USA 88 9578 (1991 ), and U S Patent Nos 5,283,173, 5,667,973 5,468,614, 5,525,490, and 5,637,463 a preferred system is described in Serial Nos 09/050,863, filed March 30, 1998 and 09/359,081 filed July 22, 1999, entitled "Mammalian Protein Interaction Cloning System" For use in conjunction with these systems, a particularly useful shuttle vector is described in Serial No 09/133,944, filed August 14, 1998, entitled "Shuttle Vectors"

In general, two nucleic acids are transformed into a cell, where one is a "bait" such as the gene encoding a cell cycle protein or a portion thereof, and the otner encodes a test candidate Only if the two expression products bind to one another will an indicator, such as a fluorescent protein, be expressed Expression of the indicator indicates when a test candidate binds to the cell cycle protein and can be identified as an cell cycle protein Using the same system and the identified cell cycle proteins the reverse can be performed Namely, the cell cycle proteins provided herein can be used to identify new baits, or agents which interact with cell cycle proteins Additionally, the two-hybrid system can be used wherein a test candidate is added in addition to the bait and the cell cycle protein encoding nucleic acids to determine agents which interfere with the bait, such as lAPs or p53, and the cell cycle protein

In one embodiment, a mammalian two-hybπd system is preferred Mammalian systems provide post-translational modifications of proteins which may contribute significantly to their ability to interact In addition, a mammalian two-hybrid system can be used in a wide variety of mammalian cell types to mimic the regulation induction, processing, etc of specific proteins within a particular cell type For example, proteins involved in a disease state (i e , cancer, apoptosis related disorders) could be tested in the relevant disease cells Similarly, for testing of random proteins, assaying them under the relevant cellular conditions will give the highest positive results Furthermore, the mammalian cells can be tested under a variety of experimental conditions that may affect intracellular protein-protein interactions, such as in the presence of hormones, drugs, growth factors and cytokmes, radiation, chemotherapeutics, cellular and chemical stimuli, etc , that may contribute to conditions which can effect protein-protein interactions, particularly those involved in cancer

Assays involving binding such as the two-hybrid system may take into account non-specific binding proteins (NSB)

Expression in various cell types, and assays for cell cycle activity are described above The activity assays, such as having an effect on lAPs binding or p53 activation can be performed to confirm the activity of cell cycle proteins which have already been identified by their sequence identity/similarity or binding to at least one lAPs as well as to further confirm the activity of lead compounds identified as modulators of ING2

The components provided herein for the assays provided herein may also be combined to form kits. The kits can be based on the use of the protein and/or the nucleic acid encoding the cell cycle proteins In one embodiment, other components are provided in the kit Such components include one or more of packaging, instructions, antibodies, and labels Additional assays such as those used in diagnostics are further described below

In this way, bioactive agents are identified Compounds with pharmacological activity are able to enhance or interfere with the activity of the cell cycle protein The compounds having the desired pharmacological activity may be administered in a physiologically acceptable earner to a host, as further described below

The present discovery relating to the role of cell cycle proteins in the cell cycle thus provides methods for inducing or preventing cell proliferation in cells In a preferred embodiment, the cell cycle proteins, and particularly cell cycle protein fragments, are useful in the study or treatment of conditions which are mediated by the cell cycle proteins, i e to diagnose, treat or prevent cell cycle associated disorders Thus, "cell cycle associated disorders" or "disease state" include conditions involving both insufficient or excessive cell proliferation, and preferably cancer

Thus, in one embodiment, cell cycle regulation in cells or organisms are provided In one embodiment, the methods comprise administering to a cell or individual in need thereof, a cell cycle protein in a therapeutic amount Alternatively, an anti-cell cycle antibody that reduces or eliminates the biological activity of the endogeneous cell cycle protein is administered In another embodiment, a bioactive agent as identified by the methods provided herein is administered Alternatively, the methods comprise administering to a cell or individual a recombinant nucleic acid encoding an cell cycle protein As will be appreciated by those in the art, this may be accomplished in any number of ways In a preferred embodiment, the activity of cell cycle is increased by increasing the amount of cell cycle in the ceil for example by overexpressmg the endogeneous cell cycle or by administering a gene encoding a cell cycle protein using known gene-therapy techniques, for example In a preferred embodiment, the gene therapy techniques include the incorporation of the exogeneous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated by reference in its entirety

Without being bound by theory, it appears that cell cycle protein is an important protein in the cell cycle Accordingly, disorders based on mutant or variant cell cycle genes may be determined In one embodiment, the invention provides methods for identifying cells containing variant cell cycle genes compπsing determining all or part of the sequence of at least one endogeneous cell cycle genes in a cell As will be appreciated by those in the art, this may be done using any number of sequencing techniques In a preferred embodiment, the invention provides methods of identifying the cell cycle genotype of an individual comprising determining all or part of the sequence of at least one cell cycle gene of the individual This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue The method may include comparing the sequence of the sequenced cell cycle gene to a known cell cycle gene, i e a wild-type gene

The sequence of all or part of the cell cycle gene can then be compared to the sequence of a known cell cycle gene to determine if any differences exist This can be done using any number of known sequence identity programs, such as Bestfit, etc In a preferred embodiment, the presence of a difference in the sequence between the cell cycle gene of the patient and the known cell cycle gene is indicative of a disease state or a propensity for a disease state

In one embodiment, the invention provides methods for diagnosing a cell cycle related condition in an individual The methods comprise measuring the activity of cell cycle in a tissue from the individual or patient, which may include a measurement of the amount or specific activity of a cell cycle protein This activity is compared to the activity of cell cycle from either a unaffected second individual or from an unaffected tissue from the first individual When these activities are different, the first individual may be at risk for a cell cycle associated disorder In this way, for example, monitoring of various disease conditions may be done, by monitoring the levels of the protein or the expression of mRNA therefor Similarly, expression levels may correlate to the prognosis

In one aspect, the expression levels of cell cycle protein genes are determined in different patient samples or cells for which either diagnosis or prognosis information is desired Gene expression monitoring is done on genes encoding cell cycle proteins In one aspect the exDression levels of cell cycle protein genes are determined for different cellular states, such as normal celis and ceils undergoing apoptosis or transformation By comparing cell cycle protein gene expression levels in ceils in different states, information including both up- and down-regulation of cell cycle protein genes is obtained, which can be used in a number of ways For example, the evaluation of a particular treatment regime may be evaluated does a chemotherapeutic drug act to improve the long-term prognosis in a particular patient Similarly, diagnosis may be done or confirmed by comparing patient samples Furthermore, these gene expression levels allow screening of drug candidates with an eye to mimicking or altering a particular expression level This may be done by making biochips comprising sets of important cell cycle protein genes, such as those of the present invention, which can then be used in these screens These methods can also be done on the protein basis, that is, protein expression levels of the cell cycle proteins can be evaluated for diagnostic purposes or to screen candidate agents In addition, the cell cycle protein nucleic acid sequences can be administered for gene therapy purposes, including the administration of antisense nucleic acids, or the cell cycle proteins administered as therapeutic drugs

Cell cycle protein sequences bound to biochips include both nucleic acid and ammo acid sequences as defined above In a preferred embodiment, nucleic acid probes to cell cycle protein nucleic acids (both the nucleic acid sequences having the sequences outlined in the Figures and/or the complements thereof) are made The nucleic acid probes attached to the biochip are designed to be substantially complementary to the cell cycle protein nucleic acids, i e the target sequence (either the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs As outlined below, this complementarity need not be perfect, there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence Thus, by "substantially complementary" herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions, particularly high stringency conditions, as outlined herein

A "nucleic acid probe" is generally single stranded but can be partially single and partially double stranded The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence In general, the nucleic acid probes range from about 8 to about 100 bases long, with from about 10 to about 80 bases being preferred, and from about 30 to about 50 bases being particularly preferred In some embodiments much longer nucleic acids can be used, up to hundreds of bases (e g , whole genes)

As will be appreciated by those in the art, nucleic acids can be attached or immobilized to a solid support in a wide variety of ways By "immobilized' and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below The binding can be covalent or non-covalent By "non-covalent binding" and grammatical equivalents herein is meant one or more of either electrostatic, hydrophilic, and hydrophobic interactions Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidm to the support and the non-covalent binding of the biotmylated probe to the streptavidm. By "covalent binding" and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules Immobilization may also involve a combination of covalent and non- covalent interactions

In general, the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art As descπbed herein, the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip

The biochip comprises a suitable solid substrate. By "substrate" or "solid support" or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method As will be appreciated by those in the art, the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc ), polysacchaπdes, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc In general, the substrates allow optical detection and do not appreciably show fluorescence

In a preferred embodiment, the surface of the biochip and the probe may be deπvatized with chemical functional groups for subsequent attachment of the two Thus, for example, the biochip is deπvatized with a chemical functional group including, but not limited to, ammo groups, carboxy groups, oxo groups and thiol groups, with ammo groups being particularly preferred Using these functional groups, the probes can be attached using functional groups on the probes For example, nucleic acids containing ammo groups can be attached to surfaces comprising ammo groups, for example using linkers as are known in the art, for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, tecnnical section on cross-linkers, pages 155-200, incorporated herein by reference) In addition, in some cases, additional linkers, such as alkyl groups (including substituted and heteroalkyl groups) may be used

In this embodiment, oligonucleotides, corresponding to the nucleic acid probe, are synthesized as is known in the art, and then attached to the surface of the solid support As will be appreciated by those skilled in the art, either the 5' or 3' terminus may be attached to the solid support, or attachment may be via an internal nucleoside

In an additional embodiment, the immobilization to the solid support may be very strong, yet non- covalent For example, biotmylated oligonucleotides can be made, which bind to surfaces covalently coated with streptavidm, resulting in attachment

Alternatively, the oligonucleotides may be synthesized on the surface, as is known in the art For example, photoactivation techniques utilizing photopolymeπzation compounds and techniques are used In a preferred embodiment, the nucleic acids can be synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/251 16, WO 95/35505, U S Patent Nos 5,700,637 and 5,445,934, and references cited within, all of which are expressly incorporated by reference, these methods of attachment form the basis of the Affimetπx GeneChip™ technology

"Differential expression " or grammatical equivalents as used herein, refers to both qualitative as well as quantitative differences in the genes' temporal and/or cellular expression patterns within and among the cells Thus, a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, for example, normal versus apoptotic cell That is, genes may be turned on or turned off in a particular state, relative to another state As is apparent to the skilled artisan, any comparison of two or more states can be made Such a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques in one such state or cell type, but is not detectable in both Alternatively, the determination is quantitative in that expression is increased or decreased, that is, the expression of the gene is either upregulated, resulting in an increased amount of transcript, or downregulated, resulting in a decreased amount of transcript The degree to which expression differs need only be large enough to quantify via standard characterization techniques as outlined below such as by use of Affymetπx GeneChip™ expression arrays, Lockhart, Nature Biotechnology 14 1675-1680 (1996), hereby expressly incorporated by reference Other techniques include, but are not limited to, quantitative reverse transcπptase PCR, Northern analysis and RNase protection

As will be appreciated by those in the art, this may be done by evaluation at either the gene transcript, or the protein level, that is, the amount of gene expression may be monitored using nucleic acid probes to the DNA or RNA equivalent of the gene transcript, and the quantification of gene expression levels, or, alternatively, the final gene product itself (protein) can be monitored, for example through the use of antibodies to the cell cycle protein and standard immunoassays (ELISAs, etc ) or other techniques, including mass spectroscopy assays, 2D gel electrophoresis assays, etc

In another method detection of the mRNA is performed in situ In this method permeabilized cells or tissue samples are contacted with a detectably labeled nucleic acid probe for sufficient time to allow the probe to hybridize with the target mRNA Following washing to remove the non- speαfically bound probe, the label is detected For example a digoxygenm labeled πboprobe (RNA probe) that is complementary to the mRNA encoding an cell cycle protein is detected by binding the digoxygenm with an anti-digoxygen secondary antibody and developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-ιndoyl phosphate

In another preferred method, expression of cell cycle protein is performed using in situ imaging techniques employing antibodies to cell cycle proteins In this method cells are contacted with from one to many antibodies to the cell cycle proteιn(s) Following washing to remove non-specific antibody binding, the presence of the antibody or antibodies is detected In one embodiment the antibody is detected by incubating with a secondary antibody that contains a detectable label In another method the primary antibody to the cell cycle proteιn(s) contains a detectable label In another preferred embodiment each one of multiple primary antibodies contains a distinct and detectable label This method finds particular use in simultaneous screening for a plurality of cell cycle proteins The label may be detected in a fiuorometer which has the ability to detect and distinguish emissions of different wavelengths in addition, a fluorescence activated cell sorter (FACS) can be used in this method As will be appreciated by one of ordinary skill in the art, numerous other histological imaging techniques are useful in the invention and the antibodies can be used in ELISA, immunoblotting (Western blotting), i munoprecipitation, BIACORE technology, and the like

In one embodiment, the cell cycle proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to cell cycle proteins, which are useful as described herein Similarly, the cell cycle proteins can be coupled, using standard technology, to affinity chromatography columns These columns may then be used to purify cell cycle antibodies In a preferred embodiment, the antibodies are generated to epitopes unique to the ceil cycle protein, that is, the antibodies show little or no cross-reactivity to other proteins These antibodies find use in a number of applications For example, the cell cycle antibodies may be coupled to standard affinity chromatography columns and used to purify cell cycle proteins as further described below The antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the cell cycle protein

The anti-cell cycle protein antibodies may comprise polyclonal antibodies Methods of preparing polyclonal antibodies are known to the skilled artisan Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intrapeπtoneal injections The immunizing agent may include the cell cycle protein or a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanm. serum albumin, bovine thyroglobulin, and soybean trypsm inhibitor Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid a, synthetic trehalose dicorynomycoiate) The immunization protocol may be selected by one skilled in the art without undue experimentation

The anti-cell cycle protein antibodies may, alternatively, be monoclonal antibodies Monoclonal antibodies may be prepared using hybπdoma methods, such as those described by Kohler and Milstein, Nature 256 495 (1975) In a hybπdoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro

The immunizing agent will typically include the cell cycle protein or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybπdoma cell [Godmg, Monoclonal Antibodies Principles and Practice, Academic Press, (1986) pp 59-103] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed The hybπdoma ceils may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoπbosyl transferase (HGPRT or HPRT), the culture medium for the hybπdomas typically will include hypoxanthine, aminopteπn, and thymidme ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium More preferred immortalized cell lines are mui ine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol , 133 3001 (1984), Brodeur et al , Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc , New York, (1987) pp 51-63]

The culture medium in which the hybπdoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against cell cycle protein Preferably, the binding specificity of monoclonal antibodies produced by the hybπdoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme- linked immunosorbent assay (ELISA) Such techniques and assays are known in the art The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal Biochem , 107 220 (1980)

After the desired hybπdoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Godmg, supral Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium Alternatively, the hybπdoma cells may be grown in vivo as ascites in a mammal

The monoclonal antibodies secreted by the subciones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulm purification procedures such as, for example, protein a-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography

The monoclonal antibodies may also be made by recombinant DNA methods such as those described in U S Patent No 4,816,567 DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of muπne antibodies) The hybndoma cells of the invention serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulm protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous muπne sequences [U S Patent No 4,816,567, Morrison et al , supral or by covalently joining to the immunoglobulm coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody

The antibodies may be monovalent antibodies Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulm light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslmking Alternatively, the relevant cysteine residues are substituted with another am o acid residue or are deleted so as to prevent crosslmking

In vitro methods are also suitable for preparing monovalent antibodies Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art The anti-cell cycle protein antibodies of the invention may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , muπne) antibodies are chimeric immunoglobuiins, immunoglobulm chains or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulm Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity In some instances, Fv framework residues of the human immunoglobulm are replaced by corresponding non-human residues Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulm and all or substantially all of the FR regions are those of a human immunoglobulm consensus sequence The humanized antibody optimally also will comprise at least a portion of an immunoglobulm constant region (Fc), typically that of a human immunoglobulm [Jones et al , Nature, 321 522-525 (1986), Riechmann et al , Nature, 332 323-329 (1988), and Presta, Curr Op Struct Biol , 2 593- 596 (1992)]

Methods for humanizing non-human antibodies are well known in the art Generally, a humanized antibody has one or more am o acid residues introduced into it from a source which is non- human These non-human ammo acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain Humanization can be essentially performed following the method of Winter and co-workers [Jones et al , Nature, 321 522-525 (1986), Riechmann et al , Nature, 332 323-327 (1988), Verhoeyen et al , Science, 239 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody Accordingly, such "humanized" antibodies are chimeric antibodies (U S Patent No 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J Mol Biol , 227 381 (1991 ), Marks et al , J Mol Biol , 222 581 (1991 )] The techniques of Cole et al and Boerner et al are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy. Alan R Liss, p 77 (1985) and Boerner et al , J Immunol , 147(1 ) 86-95 (1991 )1 Similarly, human antibodies can be made by introducing of human immunoglobulm loci into transgenic animals, e g , mice in which the endogenous immunoglobulm genes have been partially or completely inactivated Upon challenge, human antibody production is observed which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire This approach is described for example, in U S Patent Nos 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661 ,016, and in the following scientific publications Marks et al , Bio/Technology 10, 779-783 (1992), Lonberg et al , Nature 368 856-859 (1994), Morrison, Nature 368. 812-13 (1994), Fishwild et al , Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996) Lonberg and Huszar, Intern Rev Immunol 13 65-93 (1995)

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens In the present case, one of the binding specificities is for the cell cycle protein, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit

Methods for making bispecific antibodies are known in the art Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulm heavy- chain/light-cham pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature. 305 537-539 (1983)] Because of the random assortment of immunoglobulm heavy and light chains, these hybπdomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure The purification of the correct molecule is usually accomplished by affinity chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al , EMBO J_, 0 3655- 3659 (1991 )

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulm constant domain sequences The fusion preferably is with an immunoglobulm heavy-chain constant domain comprising at least part of the hinge, CH2, and CH3 regions It is preferred to have the first heavy-chain constant region (CH1 ) containing the site necessary for light-chain binding present in at least one of the fusions DNAs encoding the immunoglobulm heavy-chain fusions and, if desired, the immunoglobulm light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism For further details of generating bispecific antibodies see for example, Suresh et al , Methods jn Enzymology, 121 210 (1986)

Heteroconjugate antibodies are also within the scope of the present invention Heteroconjugate antibodies are composed of two covalentiy joined antibodies Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U S Patent No 4 676,980], and for treatment of HIV infection [WO 91/00360, WO 92/200373, EP 03089] It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslmking agents For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond Examples of suitable reagents for this purpose include immothiolate and methyl-4-mercaptobutyπmιdate and those disclosed, for example, in U S Patent No 4,676,980

The anti-cell cycle protein antibodies of the invention have various utilities For example, anti-cell cycle protein antibodies may be used in diagnostic assays for an cell cycle protein, e g , detecting its expression in specific cells, tissues, or serum Various diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies a Manual of Techniques, CRC Press, Inc (1987) pp 147-158] The antibodies used in the diagnostic assays can be labeled with a detectable moiety The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal For example, the detectable moiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ⁵l, a fluorescent or chemiluminescent compound, such as fluorescem isothiocyanate, rhodamme, or lucifeπn, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al , Nature. 144-945 (1962), David et al , Biochemistry, 13 1014 (1974), Pain et al , J Immunol Meth , 40.219 (1981 ), and Nygren, J Histochem and Cvtochem , 30 407 (1982)

Anti-Cell cycle protein antibodies also are useful for the affinity purification of cell cycle protein from recombinant cell culture or natural sources In this process, the antibodies against cell cycle protein are immobilized on a suitable support, such a Sephadex resm or filter paper, using methods well known in the art The immobilized antibody then is contacted with a sample containing the cell cycle protein to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the cell cycle protein, which is bound to the immobilized antibody Finally, the support is washed with another suitable solvent that will release the cell cycle protein from the antibody

The anti-cell cycle protein antibodies may also be used in treatment In one embodiment, the genes encoding the antibodies are provided, such that the antibodies bind to and modulate the cell cycle protein within the cell

In one embodiment, a therapeutically effective dose of an cell cycle protein, agonist or antagonist is administered to a patient By "therapeutically effective dose" herein is meant a dose that produces the effects for which it is administered The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques As is known in the art, adjustments for cell cycle protein degradation, systemic versus localized delivery, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art A "patient" for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms Thus the methods are applicable to both human therapy and veterinary applications In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human

The administration of the cell cycle protein, agonist or antagonist of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, mtranasally, transdermally, intrapeπtonealiy, intramuscularly, intrapulmonary, vaginally, rectally, or mtraocularly In some instances for example, in the treatment of wounds and inflammation, the composition may be directly applied as a solution or spray Depending upon the manner of introduction, the compounds may be formulated in a variety of ways The concentration of therapeutically active compound in the formulation may vary from about 0 1 -100 wt %

The pharmaceutical compositions of the present invention comprise an cell cycle protein, agonist or antagonist (including antibodies and bioactive agents as described herein) in a form suitable for administration to a patient In the preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuπc acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glyco c acid, pyruvic acid, oxalic acid, maleic acid, maionic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese aluminum salts and the like Particularly preferred are the ammonium potassium, sodium, calcium, and magnesium salts Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, tπmethylamine, diethylamme, tπethylamine, tπpropylamine, and ethanolamine

The pharmaceutical compositions may also include one or more of the following carrier proteins such as serum albumin, buffers fillers such as microcrystalline cellulose, lactose, com and other starches, binding agents, sweeteners and other flavoring agents, coloring agents and polyethylene glycol Additives are well known in the art, and are used in a variety of formulations

Combinations of the compositions may be administered Moreover, the compositions may be administered in combination with other therapeutics, including growth factors or chemotherapeutics and/or radiation Targeting agents (i e ligands for receptors on cancer cells) may also be combined with the compositions provided herein

In one embodiment provided herein, the antibodies are used for immunotherapy, thus, methods of immunotherapy are provided By "immunotherapy" is meant treatment of cell cycle protein related disorders with an antibody raised against a cell cycle protein As used herein, immunotherapy can be passive or active Passive immunotherapy, as defined herein, is the passive transfer of antibody to a recipient (patient) Active immunization is the induction of antibody and/or T-cell responses in a recipient (patient) Induction of an immune response can be the consequence of providing the recipient with an cell cycle protein antigen to which antibodies are raised As appreciated by one of ordinary skill in the art, the cell cycle protein antigen may be provided by injecting an cell cycle protein against which antibodies are desired to be raised into a recipient, or contacting the recipient with an cell cycle protein nucleic acid, capable of expressing the cell cycle protein antigen, under conditions for expression of the cell cycle protein antigen

In a preferred embodiment, a therapeutic compound is conjugated to an antibody, preferably an cell cycle protein antibody The therapeutic compound may be a cytotoxic agent In this method, targeting the cytotoxic agent to apoptotic cells or tumor tissue or cells, results in a reduction in the number of afflicted cells, thereby reducing symptoms associated with apoptosis, cancer cell cycle protein related disorders Cytotoxic agents are numerous and varied and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins Suitable toxins and their corresponding fragments include diptheπa A chain, exotoxm A chain, πcin A chain, abπn A chain, curcm, crotm, phenomycm, enomycm and the like Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against cell cycle proteins, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody

In a preferred embodiment, cell cycle protein genes are administered as DNA vaccines, either single nucleic acids or combinations of cell cycle protein genes Naked DNA vaccines are generally known in the art, see Brower, Nature Biotechnology 16 1304-1305 (1998) Methods for the use of nucleic acids as DNA vaccines are well known to one of ordinary skill in the art, and include placing an ceil cycle protein gene or portion of an cell cycle protein nucleic acid under the control of a promoter for expression in a patient The cell cycle protein gene used for DNA vaccines can encode full-length cell cycle proteins, but more preferably encodes portions of the cell cycle proteins including peptides derived from the cell cycle protein In a preferred embodiment a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a cell cycle protein gene Similarly, it is possible to immunize a patient with a plurality of cell cycle protein genes or portions thereof, as defined herein Without being bound by theory, following expression of the polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helper T- cells and antibodies are induced which recognize and destroy or eliminate cells expressing cell cycle proteins

53 In a preferred embodiment, the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine Such adjuvant molecules include cytokines that increase the immunogenic response to the cell cycle protein encoded by the DNA vaccine. Additional or alternative adjuvants are known to those of ordinary skill in the art and find use in the invention.

The examples described above serve to more fully describe the manner of using the above- described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All references cited herein are expressly incorporated by reference in their entirety. Moreover, all sequences displayed, cited by reference or accession number in the references are incorporated by reference herein

54

Claims

CLAIMS We claim

1 A recombinant nucleic acid encoding a cell cycle protein comprising a nucleic acid that hybridizes under high stringency conditions to a sequence complementary to that set forth in Figure 1 , 3, 5, 7, or 9

2 The recombinant nucleic acid of claim 1 wherein said protein binds to an lAPs

3 The recombinant nucleic acid of claim 1 comprising a nucleic acid sequence as set forth in Figure 1 , 3, 5, 7, or 9

4 A recombinant nucleic acid encoding a cell cycle protein comprising a nucleic acid having at least 85% sequence identity to a sequence as set forth in Figure 1 , 3, 5, 7, or 9

5 A recombinant nucleic acid encoding an ammo acid sequence as shown in Figure 2, 4, 6, 8, or 10

6 An expression vector comprising the recombinant nucleic acid according to any one of claims 1 , 2, 3, 4, or 5, operably linked to regulatory sequences recognized by a host cell transformed with the nucleic acid

7 A host cell comprising the recombinant nucleic acid according to any one of claims 1 , 2, 3, 4, or 5

8 A host cell comprising the vector of claim 6

9 A process for producing a cell cycle protein comprising cultuπng the host cell of claim 7 or 8 under conditions suitable for expression of a cell cycle protein

10 A process according to claim 9 further comprising recovering said cell cycle protein

11 A recombinant cell cycle protein encoded by the nucleic acid of any of claims 1 , 2, 3, 4, or 5

12 A recombinant polypeptide comprising an ammo acid sequence having at least 80% sequence identity with the sequence set forth in Figure 2, 4, 6, 8, or 10

13 The recombinant polypeptide of claim 12 wherein said polypeptide binds to an lAPs

14 The recombinant polypeptide of claim 12 wherein said sequence is set forth in Figure 2, 4, 6, 8, or 10

15 An isolated polypeptide which specifically binds to a cell cycle protein according to claim 13

16 A polypeptide according to claim 15 that is an antibody

17 A polypeptide according to claim 16 wherein said antibody is a monoclonal antibody

18 The monoclonal antibody of claim 17 wherein said antibody reduces or eliminates the biological function of said cell cycle protein

19 A method for screening for a bioactive agent capable of binding to a cell cycle protein, said method comprising a) combining a cell cycle protein and a candidate bioactive agent, and b) determining the binding of said candidate bioactive agent to said cell cycle protein

20 A method for screening for a bioactive agent capable of interfering with the binding of a cell cycle protein and lAPs, said method comprising a) combining a cell cycle protein, a candidate bioactive agent and an lAPs, and b) determining the binding of said cell cycle protein and said lAPs

21 A method according to Claim 20, wherein said cell cycle protein and said lAPs are combined first

22 A method for screening for a bioactive agent capable of modulating the activity of cell cycle protein, said method comprising a) adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding a cell cycle protein, and b) determining the effect of said candidate bioactive agent on said cell

23 A method according to Claim 22, wherein a library of candidate bioactive agents is added to a plurality of cells comprising a recombinant nucleic acid encoding a cell cycle protein

24 A method of modulating tumor growth comprising administering ING2 in an effective amount

25 The method of claim 24 wherein p53 is also administered