WO1997046679A1 - Variantes de cycline-c, leurs utilisations diagnostiques et therapeutiques - Google Patents

Variantes de cycline-c, leurs utilisations diagnostiques et therapeutiques Download PDF

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WO1997046679A1
WO1997046679A1 PCT/US1997/009709 US9709709W WO9746679A1 WO 1997046679 A1 WO1997046679 A1 WO 1997046679A1 US 9709709 W US9709709 W US 9709709W WO 9746679 A1 WO9746679 A1 WO 9746679A1
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cyclin
amino acid
seq
cell
acid polymer
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PCT/US1997/009709
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WO1997046679A9 (fr
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Jill M. Lahti
Vincent J. Kidd
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St. Jude Children's Research Hospital
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Publication of WO1997046679A9 publication Critical patent/WO1997046679A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • This invention relates generally to naturally occurring alternative forms of a protein that interacts with a protein kinase. This interaction has been proposed to be involved in the regulation of RNA transcription.
  • the invention more particularly relates to a truncated form of the protein, a more stable form of the protein, related biomolecules and to methods of making and using the same, including diagnostic and therapeutic uses.
  • the invention further includes corresponding amino acid and nucleotide sequences.
  • the cell cycle for replicating cells can be divided into two periods: (1) the cell division period, when the cell divides and separates, with each daughter cell receiving identical copies of the DNA; and (2) the period of growth, known as the interphase period.
  • the cell division period is termed the M (mitotic) period.
  • the interphase period in eucaryotes is further divided into three successive phases: GI (gap 1) phase, which directly follows the M period; S (synthetic) phase, which follows GI ; and G2 (gap 2) phase, which follows the S phase, and immediately precedes the M period.
  • GI gap 1
  • S synthetic phase
  • G2 G2 phase
  • the cell passes a restrictive point and becomes committed to duplicate its DNA. At this point, the cell is also committed to divide.
  • the cell replicates DNA. The net result is that during the G2 phase, the cell contains two copies of all of the DNA present in the GI phase.
  • the cells divide with each daughter cell receiving identical copies of the DNA. Each daughter cell starts the next round of the growth cycle by entering the GI phase.
  • the GI phase represents the interval in which cells respond maximally to extracellular signals, including mitogens, anti-proliferative factors, matrix adhesive substances, and intercellular contacts. Passage through the restrictive point late in GI phase defines the time at which cells lose their dependency on mitogenic growth factors for their subsequent passage through the cycle and, conversely, become insensitive to anti-proliferative signals induced by compounds such as transforming growth factor, cyclic AMP analogs, and rapamycin. Once past the restrictive point, cells become committed to duplicating their DNA and undergoing mitosis, as noted above, and the programs governing these processes are largely cell autonomous. (See generally, Darnell et al. (1986) in Molecular Cell Biology, pp. 146-148, Scientific American Books, New York.)
  • cyclin B participates in the regulation of the G2/M transition by its association with its catalytic subunit, p34 cdc2
  • cyclin A in complexes with both p34 c c* ' and cdk2 is essential for the completion of S-phase and entry into G2-phase.
  • GI -phase cyclins Complexes formed between the D-type cyclins and either cdk4 or cdk ⁇ integrate growth factor signals and the cell cycle, allow cells to progress through GI -phase.
  • This particular cell cycle pathway is specifically altered during tumorigenesis, presumably due to its role in responses to mitogenic stimulation. Alterations have been identified in many components of this pathway, including the D-type cyclins, cyclin dependent protein kinases, and cyclin dependent kinase inhibitors (CKIs).
  • CKIs cyclin dependent kinase inhibitors
  • Another GI -phase cyclin, cyclin E, in conjunction with its catalytic subunit cdk2 appears to be essential for progression from GI -phase into S- phase and the initiation of DNA replication.
  • Cyclin E and cdk2 do not appear to be directly targeted during tumorigenesis, quite possibly due to their essential nature. (See generally, Sherr, Cell 79:551 -555 (1994)) and Sherr, Cell 73:1059-1065 (1993)).
  • CKIs cdk inhibitors
  • cell cycle brakes act to inhibit cyclin/cdk complexes by binding specifically to either the cyclin or the cdk, but generally not both.
  • CKI activity is cell cycle regulated allowing these proteins to function as inhibitors of their cognate cyclin/cdk complexes for very limited periods during the cell cycle.
  • the cdk inhibitors isolated thus far include p 21 C
  • this in ibitory activity may be conditional, and these proteins may also act to positively regulate cyclin cdk complexes by functioning as "bridge" molecules that maintain complex formation and enzyme activity.
  • the p21, p27, and p57 proteins have been found in association with multiple cyclins (including D, E, and A), while the INK4 inhibitors specifically interact with complexes containing the D-type cyclins and cdk4 or cdk6. Because these proteins are important in cell cycle control, many are targeted for inactivation by both tumor viruses and genetic alterations during oncogenesis. Loss of specific CKI function can result in unregulated DNA replication, and aid in the generation of further mutations due to accumulating DNA damage. To date, mutations and/or deletions of the pl6 INK",a gene have been observed in a broad range of tumor types, strongly supporting its role as a tumor suppressor. (See generally, Sherr et al.
  • Cyclin C was originally isolated from both human and Drosophila cDNA libraries by virtue of its ability to complement a CLN1-3 defective S. cerevisiae strain (Lew et al, Cell 66: 1197- 1206 ( 1 91 ); Leopold et al. , Cell 66: 1207- 1216 ( 1991 ); Lahue et al. , (1991 )).
  • CLN1-3 yeast cyclins function during the GI -phase of the cell cycle by helping convey external growth signals to the nucleus and promote cell cycle progression (Cross, Mol. Cell Biol.
  • cyclin C was, itself, a GI -phase cyclin regulatory partner.
  • a cdk partner for cyclin C, cdk8 indicates that, like other GI -phase cyclins, cyclin C functions to regulate a specific cdk, in a cell cycle dependent manner (Tassan et al, Proc. Natl. Acad. Sci., USA 92: 8871-8875 (1995)). Cyclin C not only associates with cdk8 in vitro, thereby activating the kinase, but also associates with cdk8 in vivo.
  • the human cdk 8 catalytic partner of cyclin C maps to human chromosome I3ql2, a region associated with the BRCA2 breast cancer susceptibility gene, but the significance of this observation is unknown.
  • the CCNC gene encoding human cyclin C has recently been cloned and localized to human chromosome 6q21 (Demetrick et al, Cytogenet. Cell Genet. 69:190-192 (1995); Li et al. (1996a)). This region of chromosome six is often deleted or altered during tumorigenesis, suggesting that the CCNC gene might be a candidate tumor suppressor (Kowalczyk et al, (1985); Prigogina et al. (1988)).
  • CCNC chronic lymphoblastic leukemias
  • ALLs acute lymphoblastic leukemias
  • SSCP single-strand conformational polymorphism
  • cyclin C functions like other cyclins, i.e., to regulate a kinase catalytic domain. It has been proposed that the cyclin C-cdk8 complex regulates RNA transcription during the cell cycle. However, to date, no factor analogous to a cdk inhibitor has been identified that, in turn, specifically regulates cyclin C/cdk8 complex activity.
  • One aspect of the present invention describes a novel amino acid polymer that specifically regulates cyclin C/cdk8 complex activity and thereby plays an important role in the regulation of RNA transcription.
  • the amino acid polymer functions in a manner that is analogous, at least in result, to other cdk inhibitors, by disabling the active cyclin C/cdk8 complex during a portion of the cell cycle.
  • the amino acid polymer functions as inhibitor of its associated protein kinase at specific times during the cell cycle.
  • the present invention includes an amino acid polymer that hinders the formation of an active cyclin C-cdk8 complex.
  • the amino acid polymer also possesses a specific binding affinity for cdk8.
  • the amino acid polymer is a protein.
  • the amino acid polymer is an active fragment of the protein.
  • the present invention includes agonists, and mimics of the amino acid polymer.
  • the amino acid polymer is a truncated form of cyclin C.
  • the amino acid polymer is a truncated form of cyclin C that comprises at least about 20% or alternatively at least about 30% of the amino acid sequence encoded by the cyclin-box region.
  • the amino acid polymer is a truncated form of cyclin C that comprises at least about 40% or alternatively at least about 50% of the amino acid sequence encoded by the cyclin-box region.
  • the amino acid polymer is a truncated form of cyclin C that comprises at least about 60% or alternatively at least about 70% of the amino acid sequence encoded by the cyclin-box region.
  • the amino acid polymer has the amino acid sequence of SEQ ID N0 4 (see, Figure IB).
  • the amino acid polymer is derived from Avian cells. In a preferred embodiment the amino acid polymer is derived from mammalian cells. In one such embodiment, the amino acid polymer is a human protein that has all of the intronic sequences removed with the exception of the introns located between exon 7 and 9 (exon 8 is included). In a particular embodiment of this type, the human amino acid polymer has an amino acid sequence of SEQ ID NO:52.
  • Another aspect of the present invention includes an isolated amino acid polymer corresponding to a human cyclin C that does not encode a PEST sequence rich carboxyterminal domain. In one such embodiment, the amino acid polymer contains additional coding sequence between sequences derived from exons 11 and 12 in the human cyclin C cDNA. In a particular embodiment, the amino acid polymer has an amino acid sequence of SEQ ID NO:50. Such an amino acid polymer can have greater stability than the native human cyclin C since the PEST sequence has been shown to be involved in the rapid turnover of many proteins.
  • a further aspect of the invention includes the use of detectable labels, such as but not limited to an enzyme, a radioactive element, a biochemiluminescent, a chromophore that absorbs in the ultraviolet and/or visible and/or infrared region of the electromagnetic spectrum; and a fluorophore.
  • detectable labels such as but not limited to an enzyme, a radioactive element, a biochemiluminescent, a chromophore that absorbs in the ultraviolet and/or visible and/or infrared region of the electromagnetic spectrum; and a fluorophore.
  • the present invention includes the amino acid polymer labeled with such a detectable label.
  • the present invention includes antibodies to all of the amino acid polymers of the instant invention.
  • there are antibodies for truncated cyclin C which have a greater affinity for the truncated protein than for the full-length form of cyclin C so that the truncated cyclin C may be distinguished from the full-length form of cyclin C.
  • the antibody is raised against a peptide comprising all or a portion of amino acids 167- 178 of SEQ ID NO:52.
  • the antibody is for an amino acid polymer of the present invention that corresponds to a human cyclin C that does not encode a PEST sequence rich carboxyterminal domain.
  • an antibody has a greater affinity for the amino acid polymer than for human cyclin C.
  • the antibody is raised against a peptide comprising all or a portion of amino acids 286- 325 of SEQ ID NO:50. 8
  • the antibodies of the present invention may be selected from polyclonal antibodies, monoclonal antibodies or chimeric (bispecific) antibodies and all such variants are considered to be included herein. Either type of antibody can further comprise a detectable label as described above.
  • a related aspect of the present invention includes an immortal cell line that produces a monoclonal antibody against the amino acid polymers of the instant invention.
  • the immortal cell line produces a monoclonal antibody against a truncated cyclin C.
  • the immortal cell line produces a monoclonal antibody against the truncated cyclin C having the amino acid sequence of SEQ ID NO:4 and/or SEQ ID NO:52.
  • the immortal cell line can produce a monoclonal antibody to SEQ ID NO:50.
  • the present invention also includes methods for isolating the amino acid polymers from bacterial, insect or animal cells.
  • nucleic acid that encode the amino acid polymers of the present application.
  • the nucleic acid is an mRNA, in other embodiments it is a recombinant DNA molecule, or a degenerate variant thereof.
  • the isolated nucleic acid encodes the amino acid sequence of SEQ ID NO:4.
  • the nucleic acid has the coding sequence for the amino acid sequence of SEQ ID NO:4 contained in SEQ ID NO:2.
  • the isolated nucleic acid encodes the amino acid sequence of SEQ ID NO:52.
  • the nucleic acid has the coding sequence for the amino acid sequence for SEQ ID NO:52 contained in SEQ ID NO:51.
  • the isolated nucleic acid encodes the amino acid sequence of SEQ ID NO:50.
  • the nucleic acid has the coding sequence for the amino acid sequence for SEQ ID NO:50 contained in SEQ ID NO:49.
  • the present invention also includes the corresponding DNAs, including cDNAs and mRNAs. Nucleic acids that hybridize to the nucleic acids of the present invention under standard and/or stringent hybridization conditions are also included in the present invention.
  • Nucleotide probes are also included in the present invention. Such nucleotide probes may be used for screening for the nucleic acids of the present invention. All of the nucleotide probes of the present invention may be labeled with a detectable label as described above.
  • the nucleotide probe comprises all or a portion of the nucleotide sequence of nucleotides 885-928 of SEQ ID NO:49. In another embodiment, the nucleotide probe comprises all or a portion of the nucleotide sequence of nucleotides 527-771 of SEQ ID NO:51. In still another embodiment, the nucleotide probe comprises all or a portion of the nucleotide sequence of SEQ ID NO:53. In still another embodiment, the nucleotide probe comprises all or a portion of the nucleotide sequence of nucleotides 357-429 of SEQ ID NO:2.
  • One aspect of the present invention includes expression vectors that contain a nucleic acid of the present invention which is operatively linked to an expression control sequence. These expression vectors may be homologously recombined in a chromosome in a cell.
  • the cell contains a nucleic acid that has been disrupted, and the cell is unable to express a functional form of the amino acid polymer of the present invention.
  • the cell is a chicken DT40 cell.
  • the cell is mammalian and can be placed into a mammalian blastocyst.
  • the blastocyst can then be re-implanted into a pseudopregnant female mammal to generate a transgenic animal.
  • the transgenic animal contains a nucleic acid that has been disrupted, and the transgenic animal cannot express a functional form of the amino acid polymer of the present invention.
  • the transgenic animal is a mouse that can be used as an animal model. When the transgenic mouse contains a disrupted nucleic acid as described above, the transgenic animal is a "knockout" mouse.
  • the present invention also includes a pharmaceutical composition for treating oncogenesis containing a therapeutically effective amount of an amino acid polymer of the present invention, or an agent capable of promoting the production and/or activity of the amino acid polymer, or an agent capable of mimicking the activity of the amino acid polymer, or an agent capable of inhibiting the production and/or activity of the amino acid polymer, and mixtures thereof.
  • An alternative aspect of the present invention includes antisense nucleic acid that functions by hybridizing to the mRNA encoding the amino acid polymers of the present invention.
  • the antisense nucleic acid is RNA.
  • the antisense nucleic acid is DNA.
  • the present invention also includes a recombinant DNA molecule having a DNA sequence which, on transcription, produces an antisense ribonucleic acid against an mRNA coding for an amino acid polymer of the present invention.
  • a ribozyme that catalyzes the cleaving of a precursor RNA as part of the process of converting the precursor RNA to an mRNA transcript encoding truncated cyclin C.
  • the precursor RNA contains the exons that encode cyclin C, and one or more introns that have one or more sites for cleaving the introns from the precursor RNA.
  • the ribozyme acts at a particular site of the precursor RNA, a site which is not cleaved during the process of converting the precursor RNA to an mRNA transcript encoding the full-length cyclin C.
  • the ribozyme is a Tetrahymena-typc ribozyme. In another embodiment the ribozyme is a Hammerhead-type ribozyme.
  • the present invention also includes a recombinant DNA molecule which, upon transcription, produces the ribozyme. In preferred embodiments the site of cleavage of the RNA precursor for the ribozyme is in the cyclin-box region.
  • Test kits are also included in the present invention.
  • Test kits contain markers with specific affinities for particular target molecules and directions for using the kit. More particularly, these test kits are for identifying target molecules which are the amino acid polymers and nucleic acids of the present invention. Markers may be labeled with the detectable labels of the present invention.
  • a nucleic acid of the present invention is identified in a biological sample using a kit containing a marker that is a detectably labeled nucleotide probe of the present invention.
  • an amino acid polymer of the present invention is identified in a biological sample using a kit containing a marker that is an antibody of the present invention.
  • the marker is an antibody that is specific for truncated cyclin C.
  • the markers are in packaged in predetermined amounts.
  • a standard target molecule known to react with the marker, is included in the kit.
  • kits may contain additional reagents that react with the marker to bestow or enhance its detectability and/or buffering materials.
  • One preferred set of directions is for using a kit to detect the presence of an amino acid polymer of the present invention by the steps of:
  • kits of the present invention to detect the presence of an alternatively spliced mRNA of the present invention by the steps of:
  • the invention also includes the full-length form of Avian cyclin C having the amino acid sequence of SEQ ID N0:3 (see. Figure IA and Figure IC) antibodies raised against it and nucleic acids that code for the amino acid sequence of SEQ ID NO:3. More specifically, the invention includes the nucleic acid having the DNA sequence of SEQ ID NO: 1 (see, Figure 1 A and Figure IC). In a related embodiment, the nucleic acid has an RNA sequence corresponding to SEQ ID NO: 1.
  • the ' invention includes the use of these biomolecules to perform targeted disruption of the cyclin C gene in chicken DT40 cells via homologous recombination. Targeted disruption is a very powerful molecular genetic tool used in drug development.
  • the chicken DT40 cell line is a valuable system for the ready isolation of mutant cells, that is technically less difficult and less time consuming than the murine model.
  • the present invention also includes methodology for distinguishing the G2/M phase (late G2 phase and M phase) from the Gl/S phase (late GI phase and S phase) in the cell cycle of a given cell. This methodology relies on the presence of the truncated cyclin C and one of the alternatively spliced mRNA e.g., CmRNAl , CmRNA2, or an mRNA-encoding the amino acid sequence of SEQ ID NO:52 during the G2/M phase and their absence during the Gl/S phase.
  • Monitoring either the alternatively spliced mRNA or truncated cyclin C alone or together may be used to distinguish these phases.
  • Directions and kits described above for identifying truncated cyclin C or the alternatively spliced mRNA may be used for this purpose.
  • the present invention also includes a method of preventing and/or treating oncogenesis by administering to an animal a therapeutically effective amount of the truncated cyclin C, an agent capable of promoting the production and/or activity of the truncated cyclin C, an agent capable of mimicking the activity of the truncated cyclin C, an agent capable of inhibiting the production of truncated cyclin C, and mixtures thereof.
  • the present invention also includes a method of promoting cell growth by administering to a subject animal a nucleic acid encoding the amino acid sequence of SEQ ID NO:50 or the amino acid polymer itself. This longer lived from of a cyclin C should be more effective since less protein will be needed to be administered or synthesized.
  • Another aspect of the invention is an in vitro method for detecting or diagnosing the presence of a disease associated with elevated or decreased levels of the truncated cyclin C in a mammalian subject having the steps of: (a) evaluating the level of the truncated cyclin C in a biological sample from a mammalian subject and (b) comparing the level detected in step (a) to a level of the truncated cyclin C present in normal subjects or in the subject at an earlier time.
  • An increase in the level of the truncated cyclin C as compared to normal levels indicates a disease associated with elevated levels of truncated cyclin C
  • decreased level of truncated cyclin C as compared to normal levels indicates a disease associated with decreased levels of truncated cyclin C.
  • a variation of this aspect of the invention tests for the presence of truncated cyclin C during the different phases of the cell cycle.
  • the presence of truncated cyclin C during the Gl/S phase or its absence during the G2/M phase is indicative of a diseased state.
  • An in vitro method for monitoring a therapeutic treatment of a disease associated with elevated or decreased levels of truncated cyclin C in a mammalian subject is also disclosed. This test evaluates the levels of the truncated cyclin C in a series of biological samples obtained at different time points from a mammalian subject undergoing a therapeutic treatment for a disease associated with elevated or decreased levels of truncated cyclin C.
  • the present invention also includes methods of delivering a recombinant DNA molecule, encoding the amino acid polymers of the present invention, to a target cell which comprises providing a virus-derived vector that has been modified to comprise the recombinant DNA molecule and causing the vector to transfect the cell.
  • virus derived vectors include but are not limited to adenovirus, adeno-associated virus and retrovirus derived vectors.
  • the recombinant DNA is designed to be transcribed in the target cell constitutively.
  • the recombinant DNA is designed to be transcribed in the target cell under regulatablc conditions.
  • the target cell is a human cell.
  • Figures 1A-1C depict nucleotide sequence(s) and predicted ORFs of the avian cyclin C and alternatively spliced cyclin C cDNAs.
  • Figure IA The avian cyclin C cDNA sequence (SEQ ID NO: 1 ) and its predicted ORF (SEQ ID NO:3). The cyclin-box region is shaded. The translational start site for the cyclin C protein is indicated by the bold M. The location of introns within the coding regions is shown by the arrowheads above the DNA sequence. The termination codon is indicated by an asterisk.
  • Figure IB The alternatively spliced exon is shown by the large, bolded arrowheads.
  • the nucleotide sequence (SEQ ID NO:2) and corresponding ORF of this alternatively spliced region is shown in bold italics.
  • the termination codons are denoted by asterisks.
  • the alternatively spliced region rejoins the normal cyclin C sequence after the second large, bolded arrowhead above the DNA sequence.
  • GenBank acquisition numbers for these sequences are as follows: cyclin C ( U40873 ), alternatively spliced cyclin C ( U40874 ).
  • Figure 1 C The predicted protein sequence of the avian cyclin C cDNA is shown in comparison to the corresponding human (line 2) and Drosophila (line 3) protein sequences.
  • Figure 2 depicts a schematic representation of the avian cyclin C gene.
  • the location of the translational start site of the cyclin C protein is shown by the ATG.
  • the termination codon for the protein is shown by the TAA. Restriction endonuclease sites are indicated above the gene structure.
  • the orientation of the gene is indicated above the schematic by 5' and 3'.
  • a comparison of the isolated cyclin C mRNAs with the positions of the exons and introns of the gene is shown below the gene.
  • the normal translational start codon shared by all of these mRNAs is indicated by the ATG.
  • Both cyclin C mRNAs 1 and 2 contain exon 4a, and, therefore contain premature in-frame termination codons indicated by the TGA and TAA.
  • Cyclin C mRNA 3 is the only mRNA capable of encoding an intact cyclin C ORF. Polyadenylation of these mRNAs is indicated by the AAA sequence.
  • GenBank accession numbers for the avian genomic cyclin C sequences are as follows: Exon 2, U40875; Exon 3, U40876; Exon 4, U40877; Exon 4a, U40878; Exon 5, U40879; Exon 6, U40880; Exon 7, U40881 ; Exon 8, U40882; Exon 9, U40883; Exon 10, U40884; Exon 1 1 , U40885; Exon 12, U40886.
  • Figures 3A-3B depict RT-PCR analysis of the various cyclin C mRNAs (1, 2. and 3; as depicted in Fig. 2) from avian cell lines and tissues. The source of the cell line or tissue is indicated above each lane. Controls include DT40 cell RNA in the absence of PCR primers (RNA only) and the chicken cyclin C cDNA (C-cDNA) as a positive control. The sizes of the RT-PCR reaction products that were sequenced are shown to the left of each panel. ( Figure 3 A) RT-PCR primers corresponding to the 5' portion of exon 4a and the 3' portion of exon 7. ( Figure 3B) RT-PCR primers corresponding to the 3' portion of exon 4a and the 5' portion of exon 2.
  • Figures 4A-4D depict Northern blot analysis of alternatively spliced cyclin C mRNA expression in synchronized DT40 cells.
  • RNA isolated from elutriated and nocodazole-blocked DT40 (B-cells) was transferred to a membrane and hybridized with either, ( Figure 4A) the alternatively spliced cyclin C PCR probe (described in Materials and Methods), ( Figure 4B) hybridization of the same RNA samples with the avian cyclin C probe, ( Figure 4C) a chicken cyclin B2 cDNA probe, or ( Figure 4D) the same RNAs stained with EtBr.
  • the location of the major alternatively spliced cyclin C transcript (1.7 kb; CmRNA-2, having SEQ ID NO:2) is shown by the arrow.
  • the location of the cyclin B2 mRNA is also appropriately indicated.
  • Figures 5A-5B depict expression of a smaller, 19 kDa cyclin C-related protein in avian cells.
  • Figure 5A To determine whether the Drosophila cyclin C antibody would recognize the avian proteins, it was used to immunoprecipitate either the full- length normal cyclin C transcript or the alternatively spliced cyclin C transcript, which produces a truncated cyclin C protein. These cyclin C proteins were generated by in vitro transcription translation (IVTT) and then immunoprecipitated as described by others (Leclerc et al., Molec. Biol Cell, 7:505-513 (1996).
  • IVTT in vitro transcription translation
  • Figure 6 shows the comparison of normal human cyclin C protein sequence and alternatively spliced form 1 (human cyclin C alternatively spliced form with an additional exon between 1 1 and 12).
  • Single letter amino acid sequence of normal human cyclin C (top line) and alternatively spliced from 1 (bottom line). Identical residues are indicated by (-). Gaps in the sequence are indicated by (/).
  • Figure 7 shows the nucleotide sequence comparison of human cyclin C and alternatively spliced human cyclin C form 1 (as defined in Figure 6).
  • the nucleotide sequence of human cyclin C is shown on the top line.
  • Alternatively spliced form 1 on the bottom line.
  • Identical nucleotides are indicated by (-) and gaps are shown by (/).
  • Figure 8 shows the comparison of normal human cyclin C protein sequence and partially spliced form 1.
  • Single letter amino acid sequence of normal human cyclin C (top line) and partially spliced form 1 (bottom line). Identical residues are indicated by (-). Gaps in the sequence are indicated by (/). The predicted termination codon is shown by *.
  • Figure 9 shows the nucleotide sequence comparison of human cyclin C and partially spliced human cyclin C form 1. The nucleotide sequence of human cyclin C is shown on the top line. Partially spliced form 1 on the bottom line. Identical nucleotides are indicated by (-) and gaps are shown by (/) and ::: indicates the location of an approximately 600 nucleotides of unsequenced DNA presumed to contain exon 8.
  • the present invention describes a novel amino acid polymer a truncated cyclin C that plays an important role in the regulation of RNA transcription by acting to attenuate the cyclin C/cdk8 complex activity.
  • the present invention includes nucleic acids that specifically encode such an amino acid polymer including two particular alternatively spliced cyclin C mRNAs (CmRNA-1 , CmRNA-2) a DNA having a nucleotide sequence of SEQ ID NO:2, and a corresponding RNA to that recombinant DNA.
  • the present invention further includes methods of making, detecting, isolating, and using the amino acid polymer as a cell cycle marker protein.
  • the present invention more specifically includes a truncated human cyclin C.
  • the human truncated cycling C has the amino acid sequence of SEQ ID NO:52.
  • the present invention includes an alternatively spliced human mRNA which encodes a cyclin C containing additional codes sequences derived from exons 1 1 and 12 in the human cyclin C cDNA.
  • the alternatively spliced human mRNA encodes an amino acid polymer having an alternative carboxy-terminal end. relative to cyclin C. This protein is more long-lived than cyclin C and may be used as a stable analog of human cyclin C.
  • Antibodies raised against the amino acid polymers of the present invention, their use for detection of the amino acid polymers of the present invention, corresponding antisense nucleic acids and ribozymes are also disclosed.
  • an amino acid polymer that binds cdk8 and hinders the cyclin C/cdk8 complex activity provides an amino acid polymer (a cyclin C/cdk8 inhibitor) that hinders the formation of an active cyclin C/cdk8 complex.
  • the amino acid polymer also has a binding affinity for cdk8.
  • the cyclin C/cdk8 inhibitor is a truncated cyclin C with a sequence set forth in SEQ ID NO:4 (see, Figure IB).
  • the truncated cyclin C has an amino acid sequence of SEQ ID NO:52.
  • hinders as used herein, is meant to encompass a mild inhibition, a complete inhibition and all intermediary states of inhibition.
  • phrases "hinders the formation of an active cyclin C-cdk8 complex" as used herein, includes inhibiting the formation of a cyclin C-cdk8 complex, by for example binding cdk8 in a competitive manner with cyclin C; inhibiting the activity of an existing cyclin C-cdk8 complex, for example by forming a tertiary complex with cyclin C and cdk8; and any combination of these two inhibitory mechanisms.
  • amino acid polymer as used herein, is used interchangeably with the term “polypeptide” and denotes a polymer comprising amino acids connected by peptide bonds.
  • cyclin C/cdk8 inhibitor is used herein to denote "the amino acid polymer" which corresponds to the "truncated cyclin C" of the present invention.
  • One such specific amino acid polymer of the invention is the truncated cyclin C having the amino acid sequence set forth in SEQ ID NO:4.
  • the "cyclin box region” extends from amino acid 9 to amino acid 175. The exact requirements for a functional cyclin box region (which is necessary for physical interaction of a cdk with an appropriate cyclin) are loosely defined by a region of shared protein sequence homology.
  • p35 has very limited sequence homology to any of the members of the cyclin gene family (i.e., cyclin A, cyclin B, cyclin C, cyclin Dl, D2, D3, cyclin E, cyclin E. and cyclin G).
  • the invention further provides an antigenic fragment of the cyclin C/cdk8 inhibitor, which can be used, e.g. , after conjugation with a carrier protein, to generate antibodies to the cyclin C/cdk8 inhibitor.
  • an antigenic fragment of the cyclin C/cdk8 inhibitor which can be used, e.g. , after conjugation with a carrier protein, to generate antibodies to the cyclin C/cdk8 inhibitor.
  • the present invention contemplates the cyclin C/cdk8 inhibitor containing synthetic amino acids, derivitized by acetylation or phosphorylation, or substituted with conservative amino acids that provide the same biochemical properties.
  • a molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor.
  • An antigenic polypeptide contains at least about 5, and preferably at least about 10, amino acids.
  • An antigenic portion of a molecule can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier molecule for immunization.
  • a molecule that is antigenic need not be itself immunogenic. i.e., capable of eliciting an immune response without a carrier.
  • Proteins having a slightly altered amino acid sequence from that described herein and presented in FIGURE IB are contemplated by the present invention. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits.
  • amino acid residues described herein are preferred to be in the "L” isomeric form and include both naturally occurring amino acids as well as amino acid analogs such as norleucine.
  • residues in the "D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxyl group present at the carboxyl terminus of a polypeptide.
  • amino acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino ⁇ terminus to carboxyl-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • amino acid polymers of the present invention may be obtained in several ways including by isolation from animal cells, by synthetic means such as solid-phase peptide synthesis or by isolation from recombinant cells that contain one or more copies of a DNA transcript encoding the cyclin C/cdk8 inhibitor.
  • polypeptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other the bonds, e.g., ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
  • Synthetic polypeptides prepared using the well known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids.
  • Amino acids used for peptide synthesis may be standard Boc (N ⁇ -amino protected N ⁇ -t-butyloxycarbonyl) amino acid resin with the standard de-protecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield (J Am. Chem. Soc. 85:2149-2154 (1963)), or the base-labile N ⁇ -amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino et al. (J. Org. Chem. 37:3403-3409 (1972)). Both Fmoc and Boc N a -amino protected amino acids can be obtained from Fluka,
  • polypeptides of the invention may comprise D-amino acids, a combination of D- and L-amino acids, and various "designer" amino acids (e.g., ⁇ -methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ - methyl amino acids, etc.) to convey special properties.
  • Synthetic amino acids include ornithine for lysine, fluorophenylalanine for phenylalanine, and norleucine for leucine or isoleucine. Additionally, by assigning specific amino acids at specific coupling steps, ⁇ -helices, ⁇ turns, ⁇ sheets, ⁇ -turns, and cyclic peptides can be generated.
  • the peptides may comprise a special amino acid at the C-terminus which incorporates either a C0 2 H or CONH 2 side chain to simulate a free glycine or a glycine-amide group. Another way to consider this special residue would be as a D or L amino acid analog with a side chain consisting of the linker or bond to the bead.
  • the pseudo-free C-terminal residue may be of the D or the L optical configuration; in another embodiment, a racemic mixture of D and L- isomers may be used.
  • the present invention further provides for determination of the structure of the amino acid polymers of the present invention, which can be provided in sufficient quantities by recombinant expression (infra) or by synthesis. This is achieved by assays based on the physical or functional properties of the product, including radioactive labeling of the product followed by analysis by gel electrophoresis, immunoassay, etc.
  • the structure of the amino acid polymers of the present invention can be analyzed by various methods known in the art. Structural analysis can be performed by identifying sequence similarity with other known proteins. The degree of similarity (or homology) can provide a basis for predicting structure and function of the cyclin C/cdk8 inhibitor, or a domain thereof. In a specific embodiment, sequence comparisons can be performed with sequences found in GenBank. using, for example, the FASTA and FASTP programs (Pearson et al. , Proc. Natl. Acad. Sci., USA 85:2444-48 (1988)).
  • the protein sequence can be further characterized by a hydrophilicity analysis (e.g., Hopp et al., Proc. Natl. Acad Sci.. USA 78:3824 (1981)).
  • a hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of the cyclin C/cdk8 inhibitor.
  • Secondary structural analysis e.g., Chou et al., Biochemistry 13:222 (1974) can also be done, to identify regions of the cyclin C/cdk8 inhibitor that assume specific secondary structures.
  • Manipulation, translation, and secondary structure prediction, as well as open reading frame prediction and plotting, can also be accomplished using computer software programs available in the art.
  • the present invention enables quantitative structural determination of cyclin C/cdk8 inhibitor, or domains thereof.
  • enough material is provided for nuclear magnetic resonance (NMR). infrared (IR), Raman, and ultraviolet (UV), and circular dichroism (CD) spectroscopic analysis.
  • NMR nuclear magnetic resonance
  • IR infrared
  • UV ultraviolet
  • CD circular dichroism
  • NMR provides very powerful structural analysis of molecules in solution, which more closely approximates their native environment (Marion et al., Biochem. Biophys. Res. Comm. 113:967-974 (1983); Bar et al., J. Magn. Reson. 65:355-360 (1985); Kimura et al, Proc. Natl. Acad.
  • co-crystals of cyclin C/cdk8 inhibitor as a complex with cdk8 and/or the cyclin C/cdk8 complex can be studied. Analysis of co-crystals provides detailed information about binding, which in turn allows for rational design of ligand agonists and antagonists. Computer modeling can also be used, especially in connection with NMR or X-ray methods (Fletterick, R. and Zoller, M. (eds.), 1986, Computer
  • Nucleic Acids Encoding the Cvclin C/cdk8 inhibitor The present invention includes isolation of mRNA encoding an cyclin C/cdk8 inhibitor factor of the invention, as well as all of the mRNAs encoding the amino acid polymers of the present invention, including naturally occurring forms of the amino acid polymers, and any antigenic fragments thereof from any animal, particularly mammalian or avian, and more particularly human, source.
  • gene refers to an assembly of nucleotides that encode a polypeptide, and includes genomic DNA nucleic acids which can contain a complete set of introns and exons, and cDNA.
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.
  • a "cassette” refers to a segment of DNA that can be inserted into a vector at specific restriction sites.
  • the segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
  • a cell has been "transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • a cell has been "transformed” by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change.
  • the transforming DNA should be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • Heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
  • the heterologous DNA includes a gene foreign to the cell.
  • nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogues thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a T m of 55°. can be used, e.g.
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40% formamide, with 5x or 6x SCC.
  • High stringency hybridization conditions correspond to the highest T m , e.g., 50% formamide, 5x or 6x SCC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art.
  • T m The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • the relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • equations for calculating T m have been derived (see Sambrook et al., supra, 9.50-0.51 ).
  • a minimum length for a hybridizable nucleic acid is at least about 18 nucleotides; preferably at least about 36 nucleotides; and more preferably the length is at least about 48 nucleotides.
  • standard hybridization conditions refers to a T m of 55 °C, and utilizes conditions as set forth above.
  • the T m is 60°C; in a more preferred embodiment, the T m is 65 °C.
  • Homologous recombination refers to the insertion of a foreign DNA sequence of a vector in a chromosome.
  • the vector targets a specific chromosomal site for homologous recombination.
  • the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • polyadenylation signals are control sequences.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
  • sequence homology in all its grammatical forms refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., Cell 50:667 (1987)).
  • sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that do not share a common evolutionary origin (see Reeck et al. , supra).
  • sequence similarity when modified with an adverb such as "highly.” may refer to sequence similarity and not a common evolutionary origin.
  • two DNA sequences are "substantially homologous" or “substantially similar” when at least about 50% (preferably at least about 75%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences.
  • Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. 1 & II, supra; Nucleic Acid Hybridization, supra.
  • two amino acid sequences are "substantially homologous” or “substantially similar” when greater than 30% of the amino acids are identical, or greater than about 60% are similar (functionally identical).
  • the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program.
  • corresponding to is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured.
  • corresponding to refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.
  • a gene encoding an amino acid polymer of the present invention can be isolated from any source, particularly from a human cDNA or genomic library. Methods for obtaining the gene are well known in the art, as described above (see, e.g., Sambrook et al., supra). Accordingly, any animal cell potentially can serve as the nucleic acid source for the molecular cloning of the gene.
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook et al., supra; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences other than those portions included by alternative splicing. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.
  • Identification of the specific DNA fragment containing the desired gene may be accomplished in a number of ways. For example, if an amount of a portion of the gene or its specific RNA, or a fragment thereof, is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to a labeled probe (Benton et al, Science 196:180 (1977); Grunstein et al, Proc. Natl. Acad. Sci., USA 72:3961 (1975)).
  • a set of oligonucleotides corresponding to the partial amino acid sequence information obtained for an amino acid polymer of the present can be prepared and used as probes for DNA encoding that protein, or as primers for cDNA or mRNA (e.g., in combination with a poly-T primer for RT-PCR).
  • a fragment is selected that is highly unique to the particular amino acid polymer. Those DNA fragments with substantial homology to the probe will hybridize.
  • the greater the degree of homology the more stringent hybridization conditions can be used.
  • high stringency hybridization conditions are used to identify an homologous mRNA for the particular mRNAs of the present invention.
  • the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product.
  • cDNA clones or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein that, e.g., has similar or identical electrophoretic migration, isoelectric focusing or non-equilibrium pH gel electrophoresis behavior, proteolytic digestion maps, or antigenic properties as known for the particular amino acid polymer.
  • the ability of a cyclin C/cdk8 inhibitor to bind to cdk8 and to hinder the active cyclin C/cdk8 complex is indicative of its identity as an cyclin C/cdk8 inhibitor of the present invention.
  • the present invention also relates to cloning vectors containing genes encoding analogs and derivatives of the amino acid polymers of the present invention, that have the same or homologous functional activity as the particular amino acid polymer, and homologs thereof from other species.
  • the production and use of derivatives and analogs related to amino acid polymers of the present invention are within the scope of the present invention.
  • the derivative or analog is functionally active, e.g., capable of exhibiting one or more functional activities associated with a wild-type cyclin C/cdk8 inhibitor of the invention.
  • Derivatives can be made by altering the encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • derivatives are made that have enhanced or increased functional activity relative to the native cyclin C/cdk8 inhibitor, or have greater stability than the amino acid polymer.
  • nucleotide coding sequences which encode substantially the same amino acid sequence as an amino acid polymer of the present invention may be used in the practice of the present invention.
  • these include but are not limited to allelic genes, homologous genes from other species, and nucleotide sequences comprising all or portions of nucleic acids encoding such amino acid polymer which arc altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change.
  • such derivatives include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of an amino acid polymer of the present invention, e.g., as set forth in SEQ ID NO:4, SEQ ID NO:50, or SEQ ID NO:52, including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution.
  • substitution of one or more amino acid residues within the sequence by an amino acid of a similar polarity, which acts as a functional equivalent may result in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine. tyrosine, asparagine. and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point.
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced a potential site for disulfide bridges with another Cys.
  • a His may be introduced as a particularly "catalytic" site (i.e.. His can act as an acid or base and is the most common amino acid in biochemical catalysis).
  • Pro may be introduced because of its particularly planar structure, which induces ⁇ -turns in the protein's structure.
  • genes encoding the amino acid polymer derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, a cloned gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • a nucleic acid sequence encoding an amino acid polymer of the present invention can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • such mutations enhance the functional activity of the mutated gene product.
  • mutagenesis Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, et al, J. Biol. Chem. 253:6551 (1978); Zoller et al, DNA 3:479-488 (1984); Oliphant et al, Gene 44: 177 (1986); Hutchinson et al, Proc. Natl. Acad. Sci., USA 83:710 (1986)), use of TAB® linkers (Pharmacia), etc.
  • PCR techniques arc preferred for site directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).
  • a truncated cyclin C fusion protein can be expressed.
  • a truncated cyclin C fusion protein comprises at least a functionally active portion of a another protein joined via a peptide bond to at least a functionally active portion of a truncated cyclin C polypeptide.
  • the sequences of the other protein can be amino- or carboxy-terminal to the truncated cyclin C sequences.
  • a recombinant DNA molecule encoding such a fusion protein comprises a sequence encoding at least a functionally active portion of the other protein joined in-frame to the truncated cyclin C coding sequence, and preferably encodes a cleavage site for a specific protease, e.g., thrombin or Factor Xa, preferably at the junction of the other protein and the truncated cyclin C.
  • the other protein is glutathione-S-transferase (GST) and the fusion protein is a GST-truncated cyclin C fusion protein that bind directly to cdk8 in vitro, including radiolabeled cdk8.
  • the other protein is green fluorescent protein (GFP) and the fusion protein is a GFP-truncated cyclin C fusion protein.
  • GFP green fluorescent protein
  • the identified and isolated gene can then be inserted into an appropriate cloning vector.
  • vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used.
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the cloned gene is contained on a shuttle vector plasmid. which provides for expansion in a cloning cell, e.g., E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired.
  • a shuttle vector which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2 ⁇ plasmid.
  • nucleotide sequence coding for an amino acid polymer of the present invention, or antigenic fragment, derivative or analog thereof, or a functionally active derivative, including a chimeric protein, thereof can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Such elements are termed herein a "promoter.”
  • a promoter a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • promoter a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • the nucleic acid encoding an amino acid polymer of the invention is operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.
  • An expression vector also preferably includes a replication origin.
  • the necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding the amino acid polymer and/or its flanking regions.
  • Potential host-vector systems include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.
  • virus e.g., vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast containing yeast vectors
  • the expression elements of vectors vary in their strengths and specificities. Depending on the host- vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • a recombinant protein of the present invention, or functional fragment, derivative, chimeric construct, or analog thereof, may be expressed chromosomally, after integration of the coding sequence by recombination.
  • any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al., 1989, supra).
  • the cell into which the recombinant vector comprising the nucleic acid encoding the amino acid polymer is cultured in an appropriate cell culture medium under conditions that provide for expression of amino acid polymer by the cell.
  • Any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination). Expression of the amino acid polymer may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
  • Promoters which may be used to control cyclin C/cdk8 inhibitor gene expression include, but are not limited to, the SV40 early promoter region (Benoist et al , Nature 290:304-310 (1981)), the promoter contained in the 3 ' long terminal repeat of Rous sarcoma virus (Yamamoto et al, Cell 22:787-797 (1981 )), the herpes thymidine kinase promoter (Wagner et al, Proc. Natl. Acad. Sci., USA 78:1441-1445 (1981)), the regulatory sequences of the metal lothionein gene (Brinster et al.
  • prokaryotic expression vectors such as the ⁇ -lactamase promoter (Villa-Kamaroff et al, Proc. Natl. Acad. Sci., USA 75:3727- 3731 (1978)), or the tac promoter (DeBoer et al Proc. Natl. Acad.
  • Vectors are introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al, J. Biol. Chem. 267:963-967 (1984); Wu et al, J. Biol. Chem. 263:14621-14624 (1988); Hartmut et al, Canadian Patent Application No. 2,012,31 1 , filed March 15, 1990).
  • truncated cyclin C or the stabilized cyclin C exemplified by the amino acid polymer having the amino acid sequence of SEQ ID NO:50 can be evaluated transgenically.
  • a transgenic mouse (or other animal) model can be used.
  • a DNA fragment encoding truncated cyclin C (hereafter referred to as the "truncated cyclin C gene") for example, can be introduced transgenically using standard techniques, either to provide for over expression of the protein, or to complement animals defective in the protein.
  • Transgenic vectors including viral vectors, or cosmid clones (or phage clones) corresponding to the wild type locus of candidate gene, can be constructed using the isolated DNA fragment encoding truncated cyclin C, as described below. Cosmids may be introduced into transgenic mice using published procedures (Jaenisch, Science 240:1468-1474 (1988)).
  • truncated cyclin C genes can be tested by examining their phenotypic effects when expressed in antisense orientation in wild-type animals. In this approach, expression of the wild-type allele is suppressed, which leads to a mutant phenotype.
  • RNA-RNA duplex formation prevents normal handling of mRNA, resulting in partial or complete elimination of wild-type gene effect. This technique has been used to inhibit TK synthesis in tissue culture and to produce phenotypes of the Kruppel mutation in Drosophila, and the Shiverer mutation in mice (Izant et al, Cell 36:1007-1015 (1984); Green et al, Annu. Rev. Biochem.
  • amino acid polymers of the present invention produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins, may be used as an immunogen to generate antibodies that recognize specific and unique portions of these polypeptides.
  • Such antibodies include but are not limited to polyclonal, monoclonal (Kohler et al , Nature 256:495-497 (1975); Kozbor et al. , Immunology Today 4:72 (1983); Cole et al. , in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
  • the antibodies of the invention may be cross reactive over species, e.g., they may recognize homologous proteins from different species. Polyclonal antibodies have greater likelihood of cross reactivity.
  • an antibody of the invention may be specific for a single form of an amino acid polymer of the present invention such an antibody specific for the human truncated cyclin C.
  • cyclin C/cdk8 inhibitor polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLFI).
  • BSA bovine serum albumin
  • KLFI keyhole limpet hemocyanin
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum bacille Calmette-Guerin
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g. , radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • radioimmunoassay e.g., ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immuno
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of an amino acid polymer of the present invention, one may assay generated hybridomas for a product which binds to the amino acid polymer fragment containing such epitope. For selection of an antibody specific to such an amino acid polymer from a particular species of animal, one can select on the basis of positive binding with the amino acid polymer expressed by or isolated from cells of that species of animal.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the amino acid polymer, e.g., for Western blotting, imaging the polypeptide in situ, measuring levels thereof in appropriate physiological samples, etc.
  • the present invention extends to the preparation of antisense nucleotides and ribozymes that may be used to interfere with the expression of the cyclin C/cdk8 inhibitor at the translational level.
  • This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.
  • Such approaches also may be used to interfere with the expression of the stabilized human cyclin C exemplified by the amino acid polymer having an amino acid sequence of SEQ ID NO:50.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (see Weintraub, 1990; Marcus-Sekura, Anal. Biochem. 172:298 (1988)). In the cell, they hybridize to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids bind mRNA and thereby interfere with the expression of the protein encoded by the mRNA.
  • Oligomers of about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into organ cells.
  • Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura, supra; Hambor et al. , J. Exp Med. 168: 1237 (1988)).
  • synthetic antisense nucleotides contain phosphoester analogs, such as phosphorothiolates, or thioesters, rather than natural phosphoester bonds. Such phosphoester bond analogs are more resistant to degradation, increasing the stability, and therefore the efficacy, of the antisense nucleic acids.
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J. Am. Med. Assoc. 260:3030 (1988)). Because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • Tetrahymena-type Tetrahymena-type and "hammerhead "-type (Hasselhoff and Gerlach, 1988).
  • Tetrahymena-typc ribozymes recognize four-base sequences, while "hammerhead "-type recognize eleven- to eighteen-base sequences. The longer the recognition sequence, the more likely it is to occur exclusively in the target mRNA species. Therefore, hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a specific mRNA species, and eighteen base recognition sequences are preferable to shorter recognition sequences.
  • Various diseases or disorders mediated by inappropriate cell cycle activity due to increased or decreased activity of the cyclin C/cdk8 inhibitor of the invention may be addressed by introducing genes that encode either antisense or ribozyme molecules that inhibit expression of the cyclin C/cdk8 inhibitor (where the disease or disorder is associated with excessive cyclin C/cdk8 inhibitor activity), or the cyclin C/cdk8 inhibitor (where the disease or disorder is associated with decreased cyclin C/cdk8 inhibitor activity).
  • in vitro or in vivo transfection with one of the foregoing genes may be useful for evaluation of cell cycle activity in an animal model, which in turn may serve for drug discovery and evaluation.
  • the present invention is directed to the treatment of tumors and other cancers by modulating the activity of a truncated cyclin C, e.g., by enhancing or inhibiting expression of the cyclin C/cdk8 inhibitor to increase or decrease its activity.
  • the invention provides for introducing an antisense nucleotide or a ribozyme specific for the alternatively spliced mRNA to inhibit truncated cyclin C activity.
  • increased expression of genes indirectly under control of truncated cyclin C may be necessary to restore appropriate cell cycle and growth characteristics to a transformed cell, in which case a transgene vector of the invention for expression of truncated cyclin C can be used.
  • control of proliferation of a cancer cell is accomplished by maintaining an active cyclin C/cdk8 complex, thereby regulating uncontrolled cell proliferation characteristic of cancer cells.
  • the present invention provides for detecting the level and activity of truncated cyclin C in cells, such as cancer cells, specifically tumor cells, the need to increase or decrease the activity of truncated cyclin C in a given cell can be readily determined.
  • dysproliferative changes are treated or prevented in epithelial tissues such as those in the cervix, esophagus, and lung.
  • epithelial tissues such as those in the cervix, esophagus, and lung.
  • the present invention provides for treatment of conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non- neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976. Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79).
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells.
  • Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder. For a review of such disorders, see Fishman et al, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985).
  • a gene for regulation of truncated cyclin C e.g., an antisense gene is introduced in vivo in a viral vector.
  • Such vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Ban * virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Ban * virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, in a specific embodiment, tumors can be specifically targeted.
  • Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSVl ) vector (Kaplitt et al , Molec.
  • an attenuated adenovirus vector such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest., 90:626-630 (1990)), and a defective adeno-associated virus vector (Samulski et al, J. Virol. 61:3096-3101 (1987); Samulski et al. J. Virol, 63:3822-3828 (1989)).
  • an appropriate immunosuppressive treatment is employed in conjunction with the viral vector, e.g.. adenovirus vector, to avoid immuno-deactivation of the viral vector and transfected cells.
  • the viral vector e.g.. adenovirus vector
  • immunosuppressive cytokines such as interleukin- 12 (IL-12), interferon- ⁇ (IFN- ⁇ ), or anti-CD4 antibody
  • IL-12 interleukin- 12
  • IFN- ⁇ interferon- ⁇
  • anti-CD4 antibody can be administered to block humoral or cellular immune responses to the viral vectors (see, e.g., Wilson, Nature Medicine (1994)).
  • a viral vector that is engineered to express a minimal number of antigens.
  • the gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153 (1983); Temin et al, U.S. Patent No. 4,650,764; Temin et al, U.S. Patent No. 4,980,289; Markowitz et al, J. Virol. 62:1 120 (1988); Temin et al. U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995, by Dougherty et al.; and Kuo et al. Blood 82:845 (1993).
  • Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995.
  • the vector can be introduced in vivo by lipofection.
  • liposomes for encapsulation and transfection of nucleic acids in vitro.
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al, Proc. Natl Acad. Sci.. USA 84:7413-7417 (1987); see Mackey et al, Proc. Natl. Acad. Sci., USA 85:8027-8031 (1988)).
  • cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner et al, Science 337:387-388 (1989)).
  • lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages.
  • Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey et al, 1988, supra).
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963-967 (1992); Wu et al, 1988, Hartmut et al, supra).
  • the diagnostic method of the present invention comprises examining a cellular sample or medium by means of an assay including an effective amount of a binding partner of the amino acid polymer, such as an anti-cyclin C/cdk8 inhibitor antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb, or oligonucleotide containing the specific sequence.
  • a binding partner of the amino acid polymer such as an anti-cyclin C/cdk8 inhibitor antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb, or oligonucleotide containing the specific sequence.
  • the present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of stimuli such as the earlier referenced polypeptide ligands, by reference to their ability to elicit the activities which are mediated by the present cyclin C/cdk8 inhibitor.
  • the cyclin C/cdk8 inhibitor can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular transcription activation activity in suspect target cells.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • a number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • An amino acid polymer of the present invention or its binding partner(s) can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3 H, l4 C, 32 P, 35 S, 36 C1, "Cr, "Co, 58 Co, 59 Fe, 9U Y, l25 I, ,3, I, and 186 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fiuorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, ⁇ -glucuronidase. ⁇ -D-glucosidase, ⁇ -D-galactosidasc, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • biosensors such as the BIAcoreTM system (Pharmacia Biosensor AB, Uppsala, Sweden), or optical immunosensor systems. These systems can be grouped into four major categories: reflection techniques; surface plasmon resonance; fiber optic techniques, and integrated optic devices. Reflection techniques include ellipsometry, multiple integral reflection spectroscopy, and fluorescent capillary fill devices. Fiber ⁇ optic techniques include evanescent field fluorescence, optical fiber capillary tube, and fiber optic fluorescence sensors. Integrated optic devices include planer evanescent field fluorescence, input grading coupler immunosensor, Mach-Zehnder interferometer, Hartman interferometer and difference interferometer sensors.
  • Holographic detection of binding reactions is accomplished detecting the presence of a holographic image that is generated at a predetermined image location when one reactant of a binding pair binds to an immobilized second reactant of the binding pair (see U.S. Patent No. 5,352,582, issued October 4, 1994 to Lichtenwalter et al).
  • Examples of optical immunosensors are described in general in a review article by G.A. Robins (Advances in Biosensors), Vol. 1, pp. 229-256, 1991. More specific description of these devices are found for example in U.S. Patents 4,810,658; 4,978,503; 5,186,897; R.A. Brady et al. (Phil. Trans. R. Soc. Land. B 316: 143-160 (1987)) and G.A. Robinson et al. (in Sensors and Actuators, Elsevier, 1992).
  • Example 1 ALTERNATIVELY SPLICED CYCLIN C mRNA IS WIDELY EXPRESSED, CELL CYCLE REGULATED, AND ENCODES A TRUNCATED CYCLIN BOX PROTEIN.
  • cyclin box region includes amino acids 9-175 as determined by homology with other cyclins i.e. , cyclin A, cyclin B, cyclin Dl , cyclin E, etc.).
  • the truncated cyclin C protein functions as endogenously encoded cyclin C inhibitor by negatively regulating cyclin C/cdk8 complex activity, in much the same way as the cyclin dependent protein kinase inhibitors function to inhibit the D-type cyclins, cyclin A and cyclin E (Sherr et al. Genes & Devel. 9: 1 149-1 163 (1995)).
  • Chicken cyclin C cDNAs are isolated from a chicken UG9 T-cell cDNA library (Lahti et l, Proc. Natl. Acad. Sci., USA 88: 10595-10960 (1991)) by low-stringency hybridization with a human cyclin C cDNA (Lew et al, supra). Twenty cDNAs are isolated from a screen of -80,000 cDNAs, and they are subsequently grouped according to size, and later, nucleotide sequence differences. DNA sequence is determined as previously described (Bunnell et al, Proc. Natl. Acad. Sci.
  • the corresponding chicken gene is isolated by screening a Cornish White Rock chicken cosmid library (Stratagene) with the full- length chicken cyclin C cDNA as previously described (Eipers et al. , Genomics 13:613-621 (1992)). EcoRI fragments containing the entire gene are subcloned into a pKS plasmid vector (Stratagene), and the resulting plasmid, as well as cosmid, DNA used for double strand DNA sequence analysis.
  • oligonucleotide primers designed for sequencing the cDNA clones all exons and intron/exon boundaries are sequenced in both directions as described (Xiang et al, supra). Oligonucleotides are spaced approximately 80-100 bp apart spanning the cDNA. All DNA sequence data is analyzed using the IntelliGenetics program.
  • oligonucleotides are used for this analysis: C-l , 5' TCCAGCAGAAGGATGCAA 3'; C-2, 5' CTCCAGCACACAGTCAAC 3'; C-3, 5' AGGTGAACATCTTAAATT 3'; C-4, 5' TAGGAGCCATTAATACTG 3'; C-5, 5' GACCACTTTGACTCAAGA 3'; C-6, 5' CTGCAGAATCACTATACA 3'; C-7, 5' GTCCATTTC ⁇ TGAACACA 3'; C-8, 5' AGCCATCTCTTTCCTCTC 3'; C-9, 5' CAAGCTAGAGCTATCATG 3'; C-10, 5' CGCTCCTTCAGTAGATCTTG 3'; C-l 1, 5 * TTGAAATTTCTGTCTGAA 3 * ; C-12.
  • RT-PCR analysis of cyclin C gene expression C-24 5' CAATGGATTTTGGATAAAC
  • RNA from chicken UG9 T-cells and DT40 B-cells is isolated by hot phenol extraction as described (Bunnell et al, supra).
  • Poly(A) + RNAs corresponding to avian liver and brain are obtained from Clonetech.
  • One microgram ( ⁇ g) of RNA is analyzed from both cell lines and tissues by reverse transcriptase - polymerase chain reactions (RT-PCR) as previously described by others (Kinzler et al.1991). Cyclin C- specific oligonucleotides are used to generate both the complementary DNA (cDNA) and the PCR products.
  • sequences of these oligonucleotides are as follows: CPP- 1 , 5' AGCGCACCTTATTGAAGCTCTT 3'; CPP-2, 5' AGGCAAGCTAG- AGCTATCATGA 3'; CPP-3, 5' AGATCTACTGAAGGAGCGCCAA 3'; CPP-4, 5' AGATTAGTTTGCCA ⁇ CAACCTACA 3'.
  • the resulting RT-PCR products are run on 1% agarose-TBE gels and the fragments excised by electroblotting, as previously described (Kinzler et al , Science 253:661 -665 ( 1991 )). The isolated fragments are then purified and ligated into the TA vector (Invitrogen). The resulting clones are screened for inserts, the plasmids purified, and double-strand DNA sequence analysis performed as described above.
  • DT40 cells are synchronized by a two-step process. Because these B-cells are normally grown in suspension culture (Buerstedde et al. Cell 67:179-188 (1991)), their sensitivity to cell cycle drugs is somewhat limited. To overcome this problem a GI -phase enriched cell population is first elutriated by centrifugal elutriation. Elutriation conditions are as follows: Cells are grown in culture to a concentration of 0.5 - 1 X 10 8 .
  • the elutriator J6- Ml centrifuge
  • the elutriator is prewashed with the same media (50% serum), and the centrifuge speed set to 2000 ⁇ m.
  • the pump is set at 8 ml/min.
  • the cell suspension is then applied to the elutriator.
  • the elutriator is washed by using the media containing 50% serum for 10 min.
  • the pump is increased from 8 ml/min to 1 1 ml/min. Cells are collected to 100 ml.
  • the collected cells are centrifuged and resuspended in regular culture media at a concentration of 0.5 X 10*' / ml.
  • the elutriated cells are analyzed by fluorescence activated cell sorting (FACS) to confirm their position in the Gl- phase of the cell cycle (Dolznig et al, Cell Growth & Differ. 6:1341-1352 (1995).
  • Nocodazole (Sigma) is then added to a final concentration of 0.5 ⁇ g/ml to block the cells for 10-12 hrs. The synchrony of these cells blockage at metaphase is again confirmed by FACS analysis.
  • the sequence of the oligonucleotides used for generating this exon 4a-specif ⁇ c probe is as follows: Primer 1 (intron 4 derived) 5'- GACTTGCTGGCTGCCTCATA- 3', Primer 2 (intron 4a derived) 5'- ATATATGAAAGGTATTACAGCCACAA-3'.
  • This probe is labeled with [ 32 P]- dCTP (NEN), the blot hybridized, washed and visualized as previously described (Lahti et al. (1991); Li et al, Gene 153: 237-242 (1995). The same blot is hybridized with a chicken cyclin B2 cDNA as a positive control (Gallant et al.
  • the alternative cDNA sequence then rejoins the predicted sequence of the normal cyclin C cDNA, but due to the 73 bp size of the insertion a frame-shift occurs in the remaining cyclin C coding region.
  • the predicted ORF of the avian cyclin C cDNA produces a 283 amino acid protein (SEQ ID NO:3) and is virtually identical to the predicted ORF of the human protein, containing only one amino acid difference at amino acid residue 1 10 of chicken (130 of human); a serine residue instead of an alanine residue ( Figure I C).
  • both the cyclin C and cyclin C-related cDNAs predict ORFs that start at a methionine residue that is identical to that used in the Drosophila cyclin C mRNA ( Figure IA and Figure IC).
  • the corresponding cyclin C gene is isolated by screening a chicken genomic cosmid library with the avian cDNA insert. Six positive cosmid clones are identified and analyzed further by restriction endonuclease mapping and DNA sequencing. The mosaic structure of the avian cyclin C gene is shown in Figure 2. The location of introns that interrupt the coding sequence in the avian gene is identical to intron positions within the human gene [ Figures 1 and 2; (Li et al, Genomics 32:253-259 (1996a))]. Intron distances in the avian cyclin C gene are established by PCR.
  • the 73 bp sequence found in the alternative cyclin C cDNA corresponds to an identical 73 bp sequence (exon 4a) located between exons 4 and 5 of the normal cyclin C gene, the same location predicted by the cyclin C-related cDNA sequence ( Figures I and 2). This sequence is not contiguous with either exons 4 or 5 in the genomic clones, indicating that it is an alternatively spliced exon located within the normal avian cyclin C gene.
  • This primer set should amplify a 360 bp region of cyclin C-related mRNA, while it would produce a > 5 kb fragment from genomic DNA due to the introns located between these exons.
  • UG9 and DT40 cell lines which are both virally-transformed tumor cell lines
  • normal liver and brain tissue poly(A + ) RNA demonstrates that all cell lines and tissues examined express the alternatively spliced 360 bp product (Figure 3A).
  • This product co- migrates with the positive control generated from the cyclin C-related cDNA and it is not found in negative controls.
  • an unknown, but prominent, band of ⁇ 600 bp was observed as well ( Figure 3 A).
  • the 360 bp and 600 bp RT-PCR fragments are excised from gels and subcloned into the transactivator (TA) vector for DNA sequence analysis.
  • TA transactivator
  • This DNA sequence analysis verifies the identity of the 360 bp product as the alternatively spliced intron in all cell lines and tissues (data not shown).
  • the larger 600 bp band contains the intronic sequence normally found between exons 4a and 5 in its entirety, but none of the intronic sequences found between the other exons included in this PCR product.
  • the size of the PCR product predicted from genomic DNA for the same region is > 4 kb.
  • the 310 bp and -1.6 kb products are observed when RT-PCR reactions are performed with the various avian cell line and tissue RNAs ( Figure 3B).
  • the size and/or secondary structure of the larger RT-PCR fragment limits the sensitivity of its detection.
  • these bands are excised and cloned into the TA vector and their respective DNA sequences are determined.
  • the smaller 310 bp fragment contains sequence from exons 2, 3, 4, and 4a, but no intronic sequences.
  • the larger 1.6 kb fragment contains these same exonic regions as well as the intronic sequence located between exons 4 and 4a.
  • RNA and protein used in these analyses are prepared from synchronized DT40 cells. Synchronous cell cycle progression of DT40 cells is achieved as described in Materials and Methods. RNA is then extracted from these cells and used for detection of the exon 4a containing cyclin C-related mRNA species with a PCR-generated probe. This probe includes only exon 4a and its immediately flanking introns, and therefore should not detect the normal, completely processed cyclin C mRNA.
  • the nature of the probe used for detecting the alternatively spliced cyclin C mRNA is important, since the alternatively spliced cyclin C mRNA varies from the normal mRNA by only 73 bp, and both species are detected as a co-migrating band when analyzed by Northern blotting with the cyclin C cDNA ( Figure 4).
  • Examination of the Northern blot probed with the exon 4a-specific probe reveals that the abundance of a major, alternatively spliced 1.7 kb cyclin C- related mRNA which varies markedly during the cell cycle, reaches maximal levels during G2/M and early GI phase of the cell cycle ( Figure 4A).
  • expression of the mRNA encoding the normal cyclin C protein is invariant during the cell cycle ( Figure 4B).
  • CmRNA-2 are comparable to the normal cyclin C mRNA only during G2/M phase, since the steady-state levels of the C mRNA-2 species diminish substantially during the remainder of the cell cycle.
  • This pattern of RNA expression is similar to that observed for cyclin B2 on the same blot ( Figure 4C).
  • Identical loading of RNA for each time point was demonstrated by EtBr staining ( Figure 4D) and ⁇ -actin hybridization.
  • the alternatively spliced cyclin C mRNA (CmRNA-2) is found to be more abundant than any of the other RNA products detected by RT-PCR containing intronic sequences (CmRNA-1 and CmRNA-3). since the major mRNA transcript is ⁇ 1.7 kb in size.
  • expression of the alternatively spliced cyclin C is cell cycle- specific and contributes to the function and/or regulation of the normal cyclin C/cdk8 complex during G2/M and early GI -phase.
  • the cell cycle regulation of the CmRNA- 2 species is definitely more dramatic than that observed for either avian or human cyclin C [ Figure 4B and (Lew et al, supra).
  • a -30 kDa cyclin C protein is detected in DT40 cell lysates from cells synchronized in G2/M-phase by nocodazole treatment (Figure 5B). These cyclin C proteins co-migrate with the IVTT products corresponding to the normal and truncated avian cyclin C cDNAs, which are detected in this experiment by Western blot analysis using the affinity purified Drosophila cyclin C antibody ( Figure 5B). Specificity of the cyclin C antibody recognition of the 19 and 30 kDa cyclin C protein species is demonstrated by competition with an avian cyclin C-GST fusion protein ( Figure 5B).
  • cyclin C gene DISCUSSION Expression of the cyclin C gene is complex.
  • two mRNAs encoding truncated cyclin box proteins are produced in a cell cycle dependent fashion.
  • a -19 kDa protein is detected by cyclin C antisera during G2/M phase, and early GI -phase but not during late GI - or S-phase, in avian cell lines and normal tissues.
  • the 19 kDa protein co-migrates with a similarly sized protein produced by in vitro transcription and translation (IVTT) of an alternatively spliced avian cyclin C- related mRNA.
  • SRB10 and SRBI 1 gene products are associated in vivo with RNA polymerase II, and this cyclin/cdk complex is essential for integrating growth regulatory signals with gene transcription (O'Neill et al, supra; Liao et al , supra; Maldonado et al, supra; Leclerc et al , supra).
  • a truncated cyclin C protein would be analogous to endogenous CKIs, binding to and inactivating cdk8 during a portion of the cell cycle.
  • p21 W ⁇ Cip ', P 27 K,P1 , and p57 Kip ⁇
  • a truncated cyclin C protein could inactivate its associated protein kinase at specific times during the cell cycle (Sherr et al, (1995)).
  • Such a truncated cyclin C protein would function as an endogenously encoded CKI that is regulated via splicing of the cognate gene.
  • CmRNA-2 encoding a CKI-like protein
  • the appearance of abundant levels of this mRNA during G2/M and early GI -phase would also support a role for the active cyclin C/cdk8 protein kinase complex during late GI - and S-phase, coincidental with the transcription of genes by activated RNA polymerase II (Akoulitchev et al, Nature 377: 557-560 (1995); Leclerc et al, supra; Maldonado et al, supra).
  • the truncated cyclin C mRNA is specifically regulated during the cell cycle and it encodes a protein. Its widespread expression, in both virally transformed cell lines and normal tissues, suggests that its function is not tumor-specific.
  • the truncated cyclin C protein functions in such a manner as to resemble other CKIs, although it is the first example of the utilization of a portion of the same cyclin protein coding region as a CKI within either the cyclin or cdk gene families.
  • GST glutathione-S-transferase
  • 6-Histidine fusion proteins containing the aminoterminal domain, or the carboxyl terminal domain specific to the full-length form of cyclin C (which will not react with the truncated cyclin C protein)
  • GST glutathione-S-transferase
  • 6-Histidine fusion proteins containing the aminoterminal domain, or the carboxyl terminal domain specific to the full-length form of cyclin C (which will not react with the truncated cyclin C protein)
  • cdk8-specific antibodies are generated by use of GST or 6-Histidine fusion constructs.
  • a human cdk8 cDNA has been obtained by RT-PCR and is analyzed by DNA sequence analysis.
  • the avian homologue is isolated and analyzed in a similar manner (Parry et al., supra; Pelech el al. , TIBS 17:233-238 ( 1992)). Rabbit polyclonal antibodies are generated and affinity purified as previously described (Parry et al, supra). Antisera's that specifically recognize the cdk8 and cyclin C proteins by both Western blotting and immunoprecipitation, using the IVTT cdk8, cyclin C, and truncated cyclin C polypeptides, are obtained and used to examine the expression and function of the truncated cyclin C protein in DT40 cells that are synchronously progressing through the cell cycle. This is an especially important point since cyclin C expression is not regulated during the cell cycle (Lew et al, supra) but the expression of the truncated cyclin C mRNA (CmRNA-2) is regulated during the cell cycle.
  • CmRNA-2 truncated cyclin C mRNA
  • Both cyclin C and cdk8 antibodies are used to analyze cellular protein kinase activity as described by Tassan et al. (supra). DT40 cells are synchronized by either nocodazole or aphidicolin block of elutriated cell fractions, as described previously.
  • the cyclin C antibodies are also used to detect similar truncated cyclin C protein made in either human or murine cell lines. Specific details of these experiments are provided below. These experiments conclusively demonstrate the existence of the truncated cyclin C protein and confirm that its expression parallels the cell cycle regulated expression of its corresponding mRNA.
  • the truncated cyclin C protein functions as an effective inhibitor of the normal cyclin C/cdk8 protein kinase complex in vitro in the Sf9 insect cell expression system.
  • the infection and/or coinfection of insect Sf9 cells with baculovirus vectors encoding human and murine cell cycle gene products has elucidated specific functional interactions between a number of cell cycle proteins (Kato et al, Genes & Devel. 7:331-342 (1993); Lee et al, Mol. Biol. Cell. 3:73-84 (1992); Parker et al, Proc. Natl. Acad. Sci., USA 89:2917-2921 (1992); Parker et al. Science 55:121 1-1214 (1995)).
  • the Sf9 expression system successfully expressed many cell cycle related products, including cyclins A, B. Dl , D2, D3 and E, and the cell cycle protein kinases cdk2, cdk4, cdk6, PCTAIRE 1 and PITSLRE ⁇ l .
  • Expression of the full-length form of cyclin C and cdk8 in these cells creates an active cyclin/cdk protein kinase complex (Kato et al (1993)). It is important to note that the avian cyclin C and truncated cyclin C proteins are highly homologous to the human cyclin C protein (99% identity for the former and at least 94% identity for the latter).
  • Baculovirus expression constructs containing the truncated cyclin C protein and cdk8, as well as versions of each of the cyclin C proteins and the cdk8 kinase containing a small portion of hemagglutinin (HA) protein sequence attached to the carboxyl-terminus are constructed by the methods used for other cyclines and cdks by Meloche et al. (Mol. Biol. Cell. 3:63-71 (1992)).
  • the cyclin C and cdk8 proteins, the (His) 6 - or HA-tagged proteins are affinity purified from nickel columns or from HA epitope affinity columns, respectively, as described by Meloche et al (supra). Additional purifications, when needed, rely upon anion- exchange and gel filtration chromatography as described by Rosenblatt et al, Proc. Natl Acad. Sci. USA 89:2824-2828 (1992), Column fractions can be assayed by SDS-PAGE and Western blotting with an appropriate antibody.
  • Fractions of cyclin C or truncated cyclin C proteins are assayed for ability to phosphorylate histone HI in the presence and absence of the cdk8 catalytic subunit. This is done by infection of Sf9 cells with each component separately (i.e., kinase or cyclin) and subsequently mixing equivalent amounts of protein from the lysates for a kinase assay, or by co- infection of Sf9 cells with multiple constructs (i.e., kinase and cyclin) and direct assay of a predetermined amount of cell lysate (Desai et al, Mol. Biol. Cell, 3:571-582 ( 1992).; Kato et al.
  • Intrinsic cellular factors are provided in the reactions by mixing mammalian cell lysates, or specific immunoprecipitated proteins containing associated factors, with insect cell lysates containing the expressed kinase and/or cyclin and assaying for kinase activity, as described (Kato et al. (1993); Matsushime et al, supra).
  • the purified cyclin and cdk produced by baculovirus infection of insect Sf9 cells are added back to total cell lysates.
  • the baculovirus encoded cyclin or cdk can be readily purified using techniques that are well- established (Kato et al. (1993).
  • the analysis of the truncated cyclin C protein function is augmented by examining the function of this protein in eukaryotic expression systems and cell lines.
  • the effect of regulated expression of the truncated cyclin C protein on the normal growth of several different mammalian cell types are studied by using the available chicken clone encoding the truncated cyclin C protein since it is identical in sequence to the human cyclin C protein for 98 of its 105 amino acid residues.
  • the cognate human homologue of the chicken mRNA is isolated.
  • the human cDNA is used in both the in vitro studies described above and the in vivo studies outlined below.
  • the preferred approach is a tightly regulated, inducible expression system. These systems permit selection of stable integrants while keeping gene expression extinguished. Thus, even if expression of either cyclin C or its truncated form is deleterious to cell viability, stable cell lines are established.
  • a human T-cell line (Jurkat) and an epithelial cell line (HeLa) are obtained that express exogenous proteins under the control of a tetracycline-responsive promoter (Resnitzky et al, Mol. Cell. Biol. 14: 1669-1679 (1994).
  • the tetracycline-responsive promoter is created by fusing the tetracycline repressor with the activation domain of VP16 (a herpes simplex viral protein), thereby creating a tetracycline-controlled transactivator (tTA) that is constitutively expressed in cells.
  • VP16 a herpes simplex viral protein
  • This tTA stimulates transcription of the gene of interest from a minimal promoter sequence containing tetracycline operator sequences, which are carried on a second plasmid.
  • This vector and its use in protein expression in human cells is described in detail by Gossen et al, Proc. Natl. Acad. Sci., USA 89:5547-5551 (1992)).
  • expression of an exogenous gene is regulated by decreasing the concentration of tetracycline in the media.
  • the tetracycline-responsive promoter expression system has been successfully used to regulate exogenously introduced GI -phase cyclins (Dl and E) in mammalian cells, as well as to regulate the developmentally controlled and tissue-specific genes in mice (Resnitzky et al, Mol Cell. Biol, 14: 1669-1679 (1994); Gu et al. Science 265: 103- 106 ( 1994)).
  • Alterations in the endogenous levels of either cyclin C or the truncated cyclin C protein result in significant changes in cell growth due to alterations of the cell cycle.
  • Enhanced expression of either of these proteins, particularly the truncated protein may also result in increased or decreased cell growth, or alternatively, programmed cell death.
  • parameters of programmed cell death can be assayed, including the appearance of apoptotic nuclei, DNA fragmentation (as sensitively assayed by fluorescently labeled nick-translated DNA (Lahti et al. (1995)), and DNA ladder formation.
  • the truncated cyclin C protein exists in humans and has a similar or identical function as the avian truncated cyclin C.
  • a human cyclin C with an alternative carboxy terminal end is identified.
  • the in vitro and in vivo systems established in this example can be used to functionally examine any genetic alterations identified in the CCNC locus in human tumors.
  • Synchronized cells are used for these experiments since expression of the alternatively spliced avian transcript, and truncated protein, is cell cycle regulated.
  • Total cell protein lysates are isolated from each fraction of cells and analyzed by Western blotting for the presence of a truncated cyclin C protein using the cyclin C amino-terminal antibody. Detection of the normal wild-type cyclin C protein provides an internal control for these experiments.
  • metabolically labeled synchronized cells are made by pulsing with [ 35 S]-methionine prior to immunoprecipitating the cyclin C proteins as described.
  • RNA and/or poly(A) + RNA from different human cell lines are converted to cDNA using cyclin C specific gene primers and then subjected to nested PCR (Li et al. ; Li et al. (1996a)).
  • cDNA are synthesized from primers originating from the 3' end of the mRNA (corresponding to exons 10-12) of the CCNC gene (which encodes cyclin C).
  • Placement of the oligonucleotides for cDNA synthesis at this position allows identification of an alternatively spliced transcript in human cells, since the alternatively spliced avian mRNA contains an insertion of an exon between exons 4 and 5 of the gene. Due to the identity of the human and avian genes (Li et al. (1996a, b), oligonucleotides for the nested PCR reactions within exons 3/4 and 5/6 are generated. The highly conserved nature of both the genes and their polypeptides, indicates that exon 4a is similar in size (73 bp) in humans.
  • oligonucleotides for the nested PCR reactions within exons 4 and 5 ensures its isolation.
  • cDNA libraries generated from RNA isolated from G2/M and early GI -phase synchronized cells are screened with the human cyclin C cDNA (Lew et al, supra; Li et al. (1996a).
  • the inserts from any resulting positive clones are analyzed by DNA sequence analysis using oligonucleotide sequencing primers located in exons 4 and 5 which are oriented towards one another.
  • Human cyclin C variant transcripts were isolated by screening a human testis cDNA library with a human cyclin C cDNA probe containing the entire coding sequence for the human cyclin C protein. In addition to the normal cyclin C cDNA. two other classes of cDNA clones have been isolated. One of these represents an alternatively spliced human mRNA. This transcript is generated by the insertion of additional coding sequence between sequences derived from exons 11 and 12 in the human cyclin C cDNA ( Figure 7). The protein encoded by this mRNA has an alternative carboxy-terminal end ( Figure 6).
  • a second class of cDNA clones represents a partially spliced mRNA. All of the intronic sequences have been removed from these transcripts with the exception of the introns located between exon 7 and 9 (exon 8 is included) ( Figure 9). Extensive analysis of other cDNA clones has revealed that this is the only partially spliced cyclin C transcript found in the cells, indicating it has biological significance. This transcript is predicted to encode a truncated cyclin C protein ( Figure 8). similar in nature although slightly larger than that found in avians.
  • CCNC human cyclin C
  • ALL acute lymphoblastic leukemia
  • Another approach for examining the functional relationship between the normal cyclin C, or the truncated cyclin C and the in vivo protein kinase catalytic subunit, cdk8, involves gene targeting in cultured vertebrate cells. Elimination of the various CCNC gene products specifically, or in combination, allow the assessment of their normal function in a manner that is distinct, but complementary, to the in vitro reconstitution and tightly regulated overexpression studies described above.
  • truncated cyclin C protein functions as an inhibitor of normal cyclin C/cdk 8 complexes is derived from studies where truncated cyclin C is specifically eliminated from vertebrate cells. Concomitantly, the effect of cyclin C deficiency or elimination is examined. Targeted disruption of a specific gene via homologous recombination has become a very powerful tool in molecular genetics (Capecchi et al. Science 244: 1288-1292 ( 1989); Doetschman et al., Proc. Natl Acad. Sci., USA 85:8583-8587)). Mouse models of disease can now be produced.
  • the approximate ratio of specifically targeted gene disruption events to random integration events after transfection of DNA constructs into mammalian cells is 1 : 10 2 to 1 : 10 5 (Serrano et al (1993)).
  • a chicken B cell line, DT40 incorporates foreign DNA by specific targeted integration at frequencies that are similar to those seen for random integration (Buerstedde et al, supra). This was not a gene-specific event, since targeted integration occurred at identically elevated frequencies at four different genetic loci (Buerstedde et al, supra)). Therefore, the chicken DT40 cell line provides a valuable system for the ready isolation of mutant cells that is technically less difficult and less time consuming than the murine model. To perform these studies the chicken homologue of the particular gene of interest needs to be isolated 71 and characterized.
  • the chicken cyclin C cDNA, truncated cyclin C cDNA, and their cognate gene have been isolated and are part of the present invention, as are polyclonal antisera that recognize both the normal and truncated cyclin C proteins by immunoprecipitation.
  • the chicken cell system described here provides a practical advantage to murine systems due to the ease of generation and decreased complexity of DT40 cell culture.
  • this cell system has already been used to eliminate both alleles of cyclin Dl and it has been demonstrated that the corresponding alteration in cell cycle progression can be evaluated.
  • This system can be used to disrupt both products of the avian CCNC gene (the normal cyclin C mRNA and the alternatively spliced mRNA encoding a truncated cyclin C protein), individually and in tandem, in the DT40 cell line to determine whether cell growth parameters and/or programmed cell death (PCD) are affected.
  • PCD programmed cell death
  • Specific disruption of cyclin C can be achieved by targeting any region of exons 5-1 1 , which contain essential portions of the cyclin C open reading frame (ORF), but are not required for expression of the truncated cyclin C protein.
  • Targeted disruption of the gene can involve the insertion of neomycin (neo), hygromycin (hyg), (j romyci ⁇ ), or (histidinol) (his) selectable marker gene cassettes into unique exonic restriction sites. These genes are derived from bacteria that are resistant to the corresponding drug.
  • antibiotic resistance genes are encoded by the expression vector containing the cyclin sequences, and are not naturally found in eucaryotic cells, those cells that survive drug treatment will contain an expression vector that also contains the cyclin gene.
  • the availability of at least four distinct selectable markers allow the generation of multiple disruption plasmids.
  • Two disruption plasmids containing either a neo or hyg cassette are prepared which target both full-length and truncated cyclin C proteins.
  • a -1 kb Eagl-Kpnl restriction fragment containing exons 1-2 is removed and replaced with the selection cassettes, resulting in the disruption of a major portion of the cyclin box domain.
  • Additional constructs are made that can target exon 4a specifically, by deletion of this exon and its immediately adjacent intronic regions using restriction enzymes that remove only this region from a large 7 kb EcoRI fragment containing exons 3 - 7.
  • disruption of only the cyclin C protein, but not the truncated cyclin C protein corresponding to the ORF encoded by exons 1 - 4a can be achieved by similar deletion of a region of the avian CCNC gene containing exon 7 by using two EcoRI fragments (-2 kb), containing exons 7 - 9, from a larger 13 kb BamHI-SacI fragment which contains exons 4 - 12 of the gene ( Figure 2).
  • These selectable marker gene cassettes have been used to disrupt the avian CCNDI gene, and are used to replace this region and disrupt the normal cyclin C protein.
  • avian CCNDI gene disruptions As performed previously for the avian CCNDI gene, targeted disruption of the CCNC gene and its encoded products are verified by Southern and Northern blotting, and by either immunoprecipitation and/or Western blotting of the protein. Single cell clones can be selected by FACS sorting, as previously performed for the avian CCNDI gene disruptions, after continuous culture in selection drug for a period of 2-3 weeks (Reznitzky et al. (1994)).
  • the ability of parental DT40, CCNC -/+, and CCNC -I- to progress normally through the cell cycle after synchronization, or undergo apoptosis in response to glucocorticoids and anti-IgM antibodies can be assessed.
  • Cell cycle studies can be performed as described for the CCNDI gene disruptions.
  • the effect of diminished cyclin C protein expression on the ability of the cells to progress through the cell cycle, as measured by BudR incorporation and mitotic indices, using both asynchronous and synchronously blocked cell populations can be examined. Further, more detailed, analyses of specific cyclin/cdk complexes can be performed.
  • the regulation of programmed cell death can be assayed as described previously (Lahti et al. (1995)).
  • DT40 cells are an early B-cell lineage that express surface IgM and can undergo programmed cell death by slgM cross-linking anti-chicken slgM antibodies (Ezhevsky et al., Mol Biol. Cell 7:553-564 (1996)).
  • slgM cross-linking anti-chicken slgM antibodies Ezhevsky et al., Mol Biol. Cell 7:553-564 (1996).
  • several different CCNC -I- clonal cell lines can be compared to one another to eliminate any effects of clonal variation.
  • RNA polymerase II protein and antibody are available commercially, and can obtained for these studies (RNA polymerase II is a well conserved protein (Akoulitchev et al, supra; Osslpow et al. Cell 83:137-146 (1995)). Generation of appropriate antibodies against avian RNA polymerase II, as described previously for both cyclin C and cdk8 can also be undertaken.
  • Immunoprecipitations and kinase assays can be performed as has been done for other cell cycle proteins. This type of analysis allows the determination of whether elimination of cyclin C leads to complete, or partial loss of cdk8 kinase activity; ultimately, these investigations can provide important information regarding the possibility of redundant cyclin, or cyclin-like, regulatory partners for this catalytic subunit in vertebrate cells, as well as its possible relationship with the cdk activating protein kinase (CAK; cdk7) (Desai et al. ( 1 . 992); Solomon et al. (1993). reviewed in Morgan (1995).
  • CAK cdk activating protein kinase
  • Analogous experimental procedures for the selection, cloning, and examination of cell cycle progression and programmed cell death parameters can be performed on targeted disruptions of the truncated cyclin C protein, as well as the simultaneous elimination of both the cyclin C and truncated cyclin C proteins, as has been described above.
  • the rescue of the phenotype by re-expressing the protein that has been eliminated can be performed.
  • a similar strategy was applied to the CCNDI gene knockouts, which successfully rescued these cells (Li et al. (1996a)).
  • SSCP single strand conformational polymorphism
  • oligonucleotides corresponding to the flanking intronic sequences immediately adjacent to this exon can be designed and used in these studies (Li et al, (1996a).
  • the presence of point mutations or other alterations in the alternatively spliced exon can be confirmed by cloning and sequencing of this region of the CCNC gene by genomic PCR.
  • mutations that might alter the ability of this exon to be normally spliced into an mRNA transcript can be examined by transient transfection analysis of appropriate plasmid expression constructs in human cells.
  • CCNC gene deletion resulting in haploinsufficiency of either the full-length and/or truncated form of the cyclin C protein.
  • the procedure involves the co-amplification of a competitive template that uses the same oligonucleotide primers as those of the target cDNA, but this competitive template can be distinguished from the target cDNA after amplification.
  • the tumor samples are examined using 3-glycerol aldehyde phosphate dehydrogenase (3-GAPH) as a positive control and pl6 IN a as a negative control.
  • hematopoietic tumors can circumvent this problem, particularly when combined with mo ⁇ hologic examination (% blast cell determinations), direct fluorescence in situ hybridization (FISH) analysis (for CCNC deletions), and cytogenetic analysis which are well known in the art (Lahti et al., (1 94)).
  • FISH direct fluorescence in situ hybridization
  • cytogenetic analysis which are well known in the art (Lahti et al., (1 94)).
  • FISH direct fluorescence in situ hybridization
  • cytogenetic analysis which are well known in the art
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • SEQUENCE DESCRIPTION SEQ ID NO:1 : AATTCGCGGC CGCCGGCCGC GGCCGGGGCA GCGGAGGGCC CGGGCCCGTC CCACGGCTCC 60
  • MOLECULE TYPE protein (ill) HYPOTHETICAL: NO (v) FRAGMENT TYPE:
  • Pro Pro Phe Met lie Ala Leu Ala Cys Leu His Val Ala Cys Val Val 195 200 205
  • HYPOTHETICAL NO (xi) SEQUENCE DESCRIPTION.
  • SEQ ID NO:42 AGCGCACCTT ATTGAAGCTC TT 22

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Abstract

L'invention concerne des ARNm de cycline C à épissage alternatif et partiel, de l'ADN recombiné et la protéine tronquée (cycline C tronquée) qu'ils codent. Les ARNm à épissage alternatif résultent de l'insertion d'exons uniques renfermant des codons à terminaison prématurée. Les ARNm à épissage partiel résultent de l'insertion d'une séquence de codage additionnel issue des exons. On démontre notamment qu'au moins l'un des ARNm de cycline C à épissage alternatif est produit en fonction d'un cycle cellulaire, comme la nouvelle protéine à séquence de cycline tronquée qu'il code. La cycline C tronquée tient lieu d'inhibiteur de cycline C à codage endogène par régulation négative de l'activité complexe cycline C/cdk8, par analogie avec les inhibiteurs de kinase de protéine dépendant de la cycline qui inhibent les cyclines de type D, la cyline A et la cycline E.
PCT/US1997/009709 1996-06-03 1997-06-03 Variantes de cycline-c, leurs utilisations diagnostiques et therapeutiques WO1997046679A1 (fr)

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Cited By (2)

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WO1999046403A1 (fr) * 1998-03-11 1999-09-16 Exonhit Therapeutics S.A. Criblage differentiel qualitatif
US6881571B1 (en) 1998-03-11 2005-04-19 Exonhit Therapeutics S.A. Qualitative differential screening

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WO1993024514A1 (fr) * 1992-05-26 1993-12-09 Mitotix Cycline de type d et utilisations correspondantes
EP0666270A2 (fr) * 1994-02-08 1995-08-09 Bristol-Myers Squibb Company Inhibiteurs peptidiques des kinases régulatrices du cycle cellulaire p33cdk2 et p34cdc2 et de l'oncoprotéine du papillomavirus E7 humain

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1993024514A1 (fr) * 1992-05-26 1993-12-09 Mitotix Cycline de type d et utilisations correspondantes
EP0666270A2 (fr) * 1994-02-08 1995-08-09 Bristol-Myers Squibb Company Inhibiteurs peptidiques des kinases régulatrices du cycle cellulaire p33cdk2 et p34cdc2 et de l'oncoprotéine du papillomavirus E7 humain

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JASKULSKI D. ET AL.: "Inhibition of cellular proliferation by antisense oligodeoxynucleotides to PCNA cyclin.", SCIENCE, vol. 240, 1988, pages 1544 - 1546, XP002039132 *
LEW D.J. ET AL.: "Isolation of three novel human cyclins by rescue of G1 cyclin (Cln) function in yeast.", CELL, vol. 66, 1991, pages 1197 - 1206, XP002039130 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999046403A1 (fr) * 1998-03-11 1999-09-16 Exonhit Therapeutics S.A. Criblage differentiel qualitatif
FR2775984A1 (fr) * 1998-03-11 1999-09-17 Bioscreen Therapeutics Sa Criblage differentiel qualitatif
US6251590B1 (en) 1998-03-11 2001-06-26 Exonhit Therapeutics S.A. Differential Qualitative screening
US6881571B1 (en) 1998-03-11 2005-04-19 Exonhit Therapeutics S.A. Qualitative differential screening
US8003375B2 (en) 1998-03-11 2011-08-23 Exonhit Therapeutics S.A. Qualitative differential screening

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