WO1993024514A1 - Cycline de type d et utilisations correspondantes - Google Patents

Cycline de type d et utilisations correspondantes Download PDF

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WO1993024514A1
WO1993024514A1 PCT/US1993/005000 US9305000W WO9324514A1 WO 1993024514 A1 WO1993024514 A1 WO 1993024514A1 US 9305000 W US9305000 W US 9305000W WO 9324514 A1 WO9324514 A1 WO 9324514A1
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leu
cyclin
ala
type
glu
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David H. Beach
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Mitotix
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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

Definitions

  • a typical cell cycle of a eukaryotic cell includes the M phase, which includes nuclear division (mitosis) and cytoplasmic division or cytokinesis and interphase, which begins with the Gl phase, proceeds into the S phase and ends with the G2 phase, which continues until mitosis begins, initiating the next phase.
  • M phase nuclear division (mitosis) and cytoplasmic division or cytokinesis and interphase
  • S phase DNA replication and histone synthesis occurs, while in the Gl and G2 phases, no net DNA synthesis occurs, although damaged DNA can be repaired.
  • restriction point or start a critical point in the Gl phase
  • a critical point or start beyond which a cell is committed to completing the S, G2 and M phases.
  • Cyclins are proteins that were discovered due to their intense synthesis following the fertilization of marine invertebrate eggs (Rosenthal, E.T. et al. , Cell 20:487 (1980) ) . It was subsequently observed that the abundance of two types of cyclin, A and B, oscillated during the early cleavage divisions due to abrupt proteolytic degradation of the polypeptides at mitosis and thus, they derived their name (Evans, T. et al . , Cell 33:389 (1983) ; Swenson, K.I. et al. , Cell 47:867 (1986); Standart, N. et al. , Dev. Biol. 124:248 (1987) ) .
  • MPF is a protein kinase in which the catalytic subunit is the frog homolog of the cdc2 protein kinase (Dunphy, W.G. et al . , Cell 54:423 (1988) ; Gautier, J. et al., Cell 54:433 (1988) ; Arion, D. et al . , Cell 55:371 (1988) ) .
  • B Three types of classes of cyclins have been identified to date: B, A and CLN cyclins.
  • the B-type cyclin has been shown to act in mitosis by serving as an integral subunit of the cdc2 protein kinase (Booher, R. et al . EMBO J. 5:3441 (1987) ; Draetta, G. et al . , Cell 55:829 (1989) ; Labbe, J.C. et al., Cell 57:253 (1989) ; Labbe, J.C. et al . , EHBO J. 8:3053 (1989) ; Meijer, L. et al . , EMBO J.
  • the A-type cyclin also independently associates with the cdc2 kinase, forming an enzyme that appears to act earlier in the division cycle than mitosis (Draetta, G. et al. , Cell 56 : 829 (1989) ; Minshull, J. et al . , EMBO J. 9:2865 (1990) ; Giordano, A. et al., Cell 58:981 (1989) ; Pines, J. et al . , Nature 345:760 (1990) ) .
  • the functional difference between these two classes of cyclins is not yet fully understood.
  • cdc2 acts at two independent points in the cell cycle: mitosis and the so-called cell cycle "start" (Hartwell, L.H., J. Mol. Biol. , 104:803 (1971) ; Nurse, P. et al, Nature 292:558 (1981) ; Piggot, J.R. et al. , Nature 298:391 (1982); Reed, S.I. et al., Proc. Nat. Acad. Sci. USA 87:5697 (1990)) .
  • the start function of the CDC28 protein also requires association of the catalytic subunit of the protein kinase with ancillary proteins that are structurally related to A and B- type cyclins.
  • This third class of cyclin has been called the Cln class, and three genes comprising a partially redundant gene family have been described (Nash, R. et al. , EMBO J. 7:4335 (1988); Hadwiger, J.A. et al., Proc. Natl . Acad. Sci. USA 85:6255 (1989) ; Richardson, H.E. et al . , Cell 55:1127 (1989)) .
  • the CLN genes are essential for execution of start and in their absence, cells become arrested in the Gl phase of the cell cycle.
  • the CLN1 and CLN2 transcripts oscillate in abundance through the cell cycle, but the CLN3 transcript does not.
  • the Cln2 protein has been shown to oscillate in parallel with its mRNA (Nash, R. et al. , EMBO J. 7:4335 (1988) ; Cross, F.R., Mol. Cell. Biol. 8:4675 (1988) ; Richardson, H.E. et al ⁇ . , Cell 59:1127 (1988) ; Wittenberg, et al. , 1990) ) .
  • cdc2 and cyclins have been found not only in embryos and yeasts, but also in somatic human cells.
  • the function of the cdc2/cyclin B enzyme appears to be the same in human cells as in other cell types (Riabowol, K. et al. , Cell 57:393 (1989)) .
  • a human A type cyclin has also been found in association with cdc2.
  • No CLN type cyclin has yet been described in mammalian cells. A better understanding of the elements involved in cell cycle regulation and of their interactions would con-tribute to a better understanding of cell replication and perhaps even alter or control . the process.
  • the present invention relates to a novel class of cyclins, referred to as D-type cyclins, which are of mammalian origin and are a new family of cyclins related to, but distinct from, previously described A, B or CLN type cyclins.
  • D-type cyclins which are of mammalian origin and are a new family of cyclins related to, but distinct from, previously described A, B or CLN type cyclins.
  • human cyclins encoded by genes shown to be able to replace a CLN-type gene essential for cell cycle start in yeast, which complement a deficiency of a protein essential for cell cycle start and which, on the basis of protein structure, are on a different branch of the evolutionary tree from A, B or CLN type cyclins.
  • Three members of the new family of D-type cyclins, referred to as the human D-type gene family, are described herein.
  • cyclin Dl or CCNDl encode small (33-34 KDa) proteins which share an average of 57% identity over the entire coding region and 78% in the cyclin box.
  • One member of this new cyclin family, cyclin Dl or CCNDl is 295 amino acid residues and has an estimated molecular weight of 33,670 daltons (Da) .
  • a second member, cyclin D2 or CCND2 is 289 amino acid residues and has an estimated molecular weight of 33,045 daltons. It has been mapped to chromosome 12p band pl3.
  • a third member, cyclin D3 or CCND3 is 292 amino acid residues and has an estimated molecular weight of approximately 32,482 daltons.
  • D-type cyclins described herein are the smallest cyclin proteins identified to date. All three cyclin genes described herein are interrupted by an intron at the same position.
  • D-type cyclins of the present invention can be produced using recombinant techniques, can be synthesized chemically or can be isolated or purified from sources in which they occur naturally.
  • the present invention includes recombinant D-type cyclins, isolated or purified D-type cyclins and synthetic D-type cyclins.
  • the present invention also relates to DNA or RNA encoding a D-type cyclin of mammalian origin, particularly of human origin, as well as to antibodies, both polyclonal and monoclonal, specific for a D-type cyclin of mammalian, particularly human, origin.
  • the present invention further relates to a method of isolating genes encoding other cyclins, such as other D-type cyclins and related (but non-D type) cyclins. It also has diagnostic and therapeutic aspects. For example, it relates to a method in which the presence and/or quantity of a D- type cyclin (or cyclins) in tissues or biological samples, such as blood, urine, feces, mucous or saliva, is determined, using a nucleic acid probe based on a D-type cyclin gene or genes ' described herein or an antibody specific for a D-type cyclin. This embodiment can be used to predict whether cells are likely to undergo cell division at an abnormally high rate (i.e.
  • the present method also relates to a diagnostic method in which the occurrence of cell division at an abnormally high rate is assessed based on abnormally high levels of a D-type cyclin(s) , a gene(s) encoding a D-type cyclin(s) or a transcription product (s) (RNA) .
  • the present invention relates to a method of modulating (decreasing or enhancing) cell division by altering the activity of at least one D-type cyclin, such as D2, D2 or D3 in cells.
  • the present invention particularly relates to a method of inhibiting increased cell division by interfering with the activity or function of a D-type cyclin(s) .
  • function of D-type cyclin (s) is blocked (totally or partially) by interfering with its ability to activate the protein kinase it would otherwise (normally) activate (e. g., p34 cdc2 or a related protein kinase) , by means of agents which interfere with D- type cyclin activity, either directly or indirectly.
  • Such agents include anti-sense sequences or other transcriptional modulators which bind D cyclin-encoding DNA or RNA; antibodies which bind either the D-type cyclin or a molecule with which a D- type cyclin must interact or bind in order to carry out its role in cell cycle start; substances which bind the D-type cyclin(s) ; agents (e.g. proteases) which degrade or otherwise inactivate the D-type cyclin(s) ; or agents (e.g., small organic molecules) which interfere with association of the D-type cyclin with the catalytic subunit of the kinase.
  • the subject invention also relates to agents
  • oligonucleotides e. g., oligonucleotides, antibodies, peptides
  • Figure 1 is a schematic representation of a genetic screen for human cyclin genes.
  • Figure 2 is the human cyclin Dl nucleic acid sequence (SEQ ID No. 1) and amino acid sequence (SEQ ID No. 2) , in which nucleotide numbers and amino acid numbers are on the right, amino acid numbers are given with the initiation methionine as number one and the stop codon is indicated by an asterisk.
  • Figure 3 is the human cyclin D2 nucleic acid sequence (SEQ ID No. 3) and amino acid sequence (SEQ ID No. 4) in which nucleotide numbers and amino acid numbers are on the right, amino acid numbers are given with the initiation methionine as number one and the stop codon is indicated by an asterisk.
  • Figure 4 is the human cyclin D3 nucleic acid sequence (SEQ ID No. 5) and amino acid sequence (SEQ ID No. 6), in which nucleotide numbers and amino acid numbers are on the right, amino acid numbers are given with the initiation methionine as number one and the stop codon is indicated by an asterisk.
  • Figure 5 shows the cyclin gene family.
  • Figure 5A shows the amino acid sequence alignment of seven cyclin genes (CYCDl-Hs, SEQ ID No. 7; CYCA-Hs, SEQ ID No. 8; CYCA-Dm, SEQ ID No. 9; CYCBl-Hs, SEQ ID No. 10; CDC13-Sp, SEQ ID No. 11; CLNl-Sc, SEQ ID No. 12; CLN3-SC, SEQ ID No. 13) , in which numbers within certain sequences indicate the number of amino acid residues omitted from the sequence as the result of insertion.
  • CYCDl-Hs SEQ ID No. 7
  • CYCA-Hs SEQ ID No. 8
  • CYCA-Dm SEQ ID No. 9
  • CYCBl-Hs SEQ ID No. 10
  • CDC13-Sp SEQ ID No. 11
  • CLNl-Sc SEQ ID No. 12
  • CLN3-SC SEQ ID No. 13
  • Figure 5B is a schematic representation of the evolutionary tree of the cyclin family, constructed using the Neighbor- Joining method; the length of horizontal line reflects the divergence.
  • Figure 6 shows alternative polyadenylation of the cyclin Dl gene transcript .
  • Figure 6A is a comparison of several cDNA clones isolated from different cell lines. Open boxes represent the 1.7 kb small transcript containing the coding region of cyclin Dl gene. Shadowed boxes represent the 3' fragment present in the 4.8 kb long transcript. Restriction sites are given above each cDNA clone to indicate the alignment of these clones.
  • Figure 6B shows the nucleotide sequence surrounding the first polyadenylation site for several cDNA clones (CYCD1- 21, SEQ ID No. 14; CYCDl-H12, SEQ ID No. 15; CYCDl-H034, SEQ ID No. 16; CYCDl-T078, SEQ ID No. 17 and a genomic clone; CYCD1-G068, SEQ ID No. 18) .
  • Figure 6C is a summary of the structure and alternative polyadenylation of the cyclin Dl gene. Open boxes represent the small transcript, the shadowed box represents the 3' sequence in the large transcript and the filled boxes indicate the coding regions.
  • Figure 7 shows the protein sequence comparison of eleven mammalian cyclins (CYCDl-Hs, SEQ ID No. 19; CYLl-Mm, SEQ ID No. 20; CYCD2-HS, SEQ ID No. 21; CYCL2-Mm, SEQ ID No. 22; CYCD3-HS, SEQ ID No. 23; CYL3-Mm, SEQ ID No. 24; CYCA-Hs, SEQ ID No. 25; CYCBl-Hs, SEQ ID No. 26; CYCB2-HS, SEQ ID No. 27; CYGC-Hs, SEQ ID No. 28; CYCE-Hs, SEQ ID No. 29) .
  • Figure 8 is a schematic representation of the genomic structure of human cyclin D genes, in which each diagram represents one restriction fragment from each cyclin D gene that has been completely sequenced. Solid boxes indicate exon sequences, open boxes indicate intron or 5' and 3' untranslated sequences and hatched boxes represent pseudogenes. The positions of certain restriction sites, ATG and stop codons are indicated at the top of each clone.
  • Figure 9 is the nucleic acid sequence (SEQ ID No. 30) and amino acid sequence (SEQ ID No. 31) of a cyclin D2 pseudogene.
  • Figure 10 is the nucleic acid sequence (SEQ ID No. 32) and the amino acid sequence (SEQ ID No. 33) of a cyclin D3 pseudogene.
  • Figure 11 is the nucleic acid sequence (SEQ ID No. 34) of 1.3 kb of human cyclin Dl promoter; the sequence ends at initiation ATG codon and transcript ion starts at approximately nucleotide -160.
  • Figure 12 is the nucleotide sequence (SEQ ID No. 35) of 1.6 kb of human cyclin D2 promoter; the sequence ends at initiation ATG codon and transcript ion starts at approximately nucleotide -170.
  • Figure 13 is the nucleotide sequence (SEQ ID No. 36) of 3.2 kb of human cyclin D3 promoter; the sequence ends at initiation ATG codon and transcription starts at approximately nucleotide -160.
  • D-type cyclins a new class of mammalian cyclin proteins, designated D-type cyclins, has been identified, isolated and shown to serve as a control element for the cell cycle start, in that they fill the role of a known cyclin protein by activating a protein kinase whose activation is essential for cell cycle start, an event in the Gl phase at which a cell becomes committed to cell division.
  • human D-type cyclin proteins, as well as the genes which encode them have been identified, isolated and shown to be able to replace CLN type cyclin known to be essential for cell cycle start in yeast.
  • the chromosomal locations of CCND2 and CCND3 have also been mapped.
  • D type cyclins
  • DNA and RNA encoding the novel D-type cyclins
  • Two yeast transformants (pCYCDl-21 and pCYCDl-19) which grew despite the lack of function of all three CLN genes and were not revertants, were identified and recovered in E. coli. Both rescued the mutant (CLN deficient) strain when reintroduced into yeast, although rescue was inefficient and the rescued strain grew relatively poorly.
  • pCYCDl-19 and pCYCDl-21 were shown, by restriction mapping and partial DNA sequence analysis, to be independent clones representing the same gene.
  • a HeLa cDNA library was screened for a full length cDNA clone, using the 1.2 kb insert of pCYCDl-21 as probe. Complete sequencing was done of the longest of nine positive clones identified in this manner (pCYCDl-H12; 1325 bp) .
  • the sequence of the 1.2 kb insert is presented in Figure 2; the predicted protein product of the gene is of approximate molecular weight 34,000 daltons.
  • Cyclin D2 and cyclin D3 cDNAs were isolated using the polymerase chain reaction and three oligonucleotide probes derived from three highly conserved regions of D-type cyclins, as described in Example 4. As described, two 5' oligonucleotides and one 3' degenerate oligonucleotide were used for this purpose.
  • the nucleotide and amino acid sequences of the CCND2 gene and encoded D2 cyclin protein are represented in Figure 3 and of the CCND3 gene and encoded D3 cyclin protein are represented in Figure 4.
  • a deposit of plasmid pCYC-D3 was made with the American Type Culture Collection (Rockville, MD) on May 14, 1991, under the terms of the Budapest Treaty.
  • cyclin Dl gene expression was studied using Northern analysis, as described in Example 2. Results showed that levels of cyclin Dl expression were very low in several cell lines. The entire coding region of the CYCDl gene was used to probe poly(A) + RNA from HeLa cells and demonstrated the presence of two major transcripts, one approximately 4.8 kb and the other approximately 1.7 kb, with the higher molecular weight form being the more abundant. Most of the cDNA clones isolated from various cDNA libraries proved to be very similar to clone _CYCD1-H12 and, thus, it appears that the 1.7 kb transcript detected in Northern blots corresponds to the nucleotide sequence of Figure 2. The origin of the larger (4.8 kb) transcript was unclear. As described in Example 2, it appears that the two mRNAs detected (4.8 kb and 1.7 kb) arose by differential polyadenylation of CYCDl ( Figure 6) .
  • cyclin Dl Differential expression of cyclin Dl in different tissues and cell lines was also assessed, as described in Example 3. Screening of cDNA libraries to obtain full length CYCDl clones had demonstrated that the cDNA library from the human glioblastoma cell line (U118 MG) used to produce yeast transformants produced many more positives than the other three cDNA libraries (human HeLa cell cDNA, human T cell cDNA, human teratocarcinoma cell cDNA) . Northern and Western blotting were carried out to determine whether cyclin Dl is differentially expressed.
  • results showed (Example 3) that the level of transcript is 7 to 10 fold higher in the glioblastoma (U118 MG) cells than in HeLa cells, and that in both HeLa and U118 MG cells, the high and low molecular weight transcripts occurred.
  • Western blotting using anti-CYLl antibody readily detected the presence of a 34kd polypeptide in the glioblastoma cells and demonstrated that the protein is far less abundant in HeLa cells and not detectable in the 293 cells.
  • the molecular weight of the anti-CYCLl cross reactive material identified in U118 MG and HeLa cells is exactly that of the human CYCDl protein expressed in E. coli.
  • results demonstrated differential occurrence of the cyclin Dl in the cell types analyzed, with the highest levels being in cells of neural origin.
  • Lambda genomic clones corresponding to the human cyclin D2 and lambda genomic clones corresponding to the human cyclin D3 were also isolated and characterized, using a similar approach.
  • One clone (XD2-G4) was shown to contain ( Figure 8B) a 2.7 kb SacI Smal fragment which includes 1620 bp of sequence 5' to the presumptive initiating methionine codon identified in D2 cDNA ( Figure 3) and a 195 bp exon followed by a 907 bp intervening sequence.
  • cyclin D a novel class of mammalian cyclins, designated cyclin D or D-type cyclin, has been identified and shown to be distinct, on the basis of structure of the gene (protein) product, from previously-identified cyclins.
  • a cDNA library carried in an appropriate yeast vector is introduced into a mutant yeast strain, such as the strain described herein (Example 1 and Experimental Procedures) .
  • the strain used contains altered CLN genes.
  • insertional mutations in the CLN1 and CLN2 genes rendered them inactive and alteration of the CLN3 gene allowed for its conditional expression from a galactose-inducible, glucose-repressible promoter; as exemplified, this promoter is a galactose- inducible, glucose-repressible promoter but others can be used.
  • Mutant yeast transformed with the cDNA library in the express ion vector are screened for their ability to grow on glucose-containing medium.
  • medium containing galactose the CLN3 gene is expressed and cell viability is maintained, despite the absence of CLN1 and CLN2.
  • medium containing glucose all CLN function is lost and the yeast cells arrest in the Gl phase of the cell cycle.
  • the ability of a yeast transformant to grow on glucose-containing medium is an indication of the presence in the transformant of DNA able to replace the function of a gene essential for cell cycle start.
  • an expression vector such as pADNS, which contains a selectable marker (the LEU2 marker is present in pADNS) .
  • Assessment of the plasmid stability shows whether the ability to grow on glucose-containing medium is the result of reversion or the presence of DNA function
  • cyclins of all types can be identified by their ability to replace CLN3 function when transformants are grown on glucose.
  • Screening of additional cDNA or genomic libraries to identify other cyclin genes can be carried out using all or a portion of the human D-type cyclin DNAs disclosed here in as probes; for example, all or a portion of the Dl, D2 or D3 cDNA sequences of Figures 2-4, respectively, or all or a portion of the corresponding genomic sequences described herein can be used as probes.
  • the hybridization conditions can be varied as desired and, as a result, the sequences identified will be of greater or lesser complementarity to the probe sequence (i.e., if higher or lower stringency conditions are used) .
  • an anti-D type cyclin antibody such as CYL1 or another raised against Dl or D3 or other human D-type cyclin, can be used to detect other recombinant D-type cyclins produced in appropriate host cells transformed with a vector containing DNA thought to encode a cyclin.
  • the PRAD1 gene which has the same sequence as the cyclin Dl gene, may play an important role in the development of various tumors (e.g., non-parathyroid neoplasia, human breast carcinomas and squamous cell carcinomas) with abnormalities in chromosome llql3.
  • tumors e.g., non-parathyroid neoplasia, human breast carcinomas and squamous cell carcinomas
  • CCND1 PRAD1
  • identification of CCND1 (PRAD1) as a candidate BCL1 oncogene provides the most direct evidence for the oncogenic potential of cyclin genes. This also suggests that other members of the D-type cyclin family may be involved in oncogenesis.
  • Region 12pl3 contains sites of several translocations that are associated with specific immunophenotypes of disease, such as acute lymphoblastic leukemia, chronic myelomoncytic leukemia, and acute myeloid leukemia.
  • the isochromosome of the short arm of chromosome 12 [l(12p)] is one of a few known consistent chromosomal abnormalities in human solid tumors and is seen in 90% of adult testicular germ cell tumors.
  • Region 6p21 has been implicated in the manifestation of chronic lymphoproliferative disorder and leiomyoma.
  • Region tp21 the locus of HLA complex, is also one of the best characterized regions of the human genome. Many diseases have been previously linked to the KLA complex, but the etiology of few of these diseases is fully understood. Molecular cloning and chromosomal localization of cyclins D2 and D3 should make it possible to determine whether they are directly involved in these translocations, and if so, whether they are activated. If they prove to be involved, diagnostic and therapeutic methods described here in can be used to assess an individual's disease state or probability of developing a condition associated with or caused by such translocations, to monitor therapy effectiveness (by assessing the effect of a drug or drugs on cell proliferation) and to provide treatment.
  • the present invention includes a diagnostic method to detect altered expression of a cyclin gene, such as cyclin Dl, D2, D3 or another D-type cyclin.
  • the method can be carried out to detect altered expression in cells or in a biological sample.
  • a cyclin gene such as cyclin Dl, D2, D3 or another D-type cyclin.
  • the method can be carried out to detect altered expression in cells or in a biological sample.
  • cyclin D genes there is high sequence similarity among cyclin D genes, which indicates that different members of D-type cyclins may use similar mechanisms in regulating the cell cycle (e.g., association with the same catalytic subunit and acting upon the same substrates) .
  • cyclin Dl is expressed differentially in tissues analyzed; in particular, it has been shown to be expressed at the highest levels in cells of neural origin (e.g., glioblastoma cells) .
  • D-type cyclin expression can be detected and/or quantitated and results used as an indicator of normal or abnormal (e.g., abnormally high rate of) cell division.
  • Differential express ion (either express ion in various cell types or of one or more of the types of D cyclins) can also be determined.
  • cells obtained from an individual are processed in order to render nucleic acid sequences in them available for hybridization with complementary nucleic acid sequences .
  • All or a portion of the Dl, D2 and/or D3 cyclin (or other D-type cyclin gene) sequences can be used as a probe (s) .
  • Such probes can be a portion of a D-type cyclin gene; such a portion must be of sufficient length to hybridize to complementary sequences in a sample and remain hybridized under the conditions used and will generally be at least six nucleotides long.
  • Hybridization is detected using known techniques (e.g., measurement of labeled hybridization complexes, if radiolabeled or fluorescently labeled oligonucleotide probed are used) .
  • the extent to which hybridization occurs is quantitated; increased levels of the D-type cyclin gene is indicative of increased potential for cell division.
  • the extent to which a D-type cyclin (or cyclins) is present in cells, in a specific cell type or in a body fluid can be determined using known techniques and an antibody specific for the D-type cyclin (s) .
  • complex formation between the D-type cyclin and the protein kinase with which it normally or typically complexes is assessed, using exogenous substrate, such as histone HI, as a substrate. Arion, D. et al . , Cell , 55:371 (1988) .
  • each diagnostic method comparison of results obtained from cells or a body fluid being analyzed with results obtained from an appropriate control (e.g., cells of the same type known to have normal D-type cyclin levels and/or activity or the same body fluid obtained from an individual known to have normal D-type cyclin levels and/or activity) is carried out.
  • Increased D-type cyclin levels and/or activity may be indicative of an increased probability of abnormal cell proliferation or oncogenesis or of the actual occurrence of abnormal proliferation or oncogenesis .
  • cyclin e.g., A, B, and/or D
  • a set of probes e.g., a set of nucleic acid probes or a set of antibodies
  • Such probes are also the subject of the present invention; they will generally be detectably labelled (e.g., with a radioactive label, a fluorescent material, biotin or another member of a binding pair or an enzyme) .
  • a method of inhibiting cell division, particularly cell division which would otherwise occur at an abnormally high rate, is also possible.
  • increased cell division is reduced or prevented by introducing into cells a drug or other agent which can block, directly or indirectly, formation of the protein kinase-D type cyclin complex and, thus, block activation of the enzyme.
  • complex formation is prevented in an indirect manner, such as by preventing transcription and/or translation of the D-type cyclin DNA and/or RNA. This can be carried out by introducing antisense oligonucleotides into cells, in which they hybridize to the cyclin-encoding nucleic acid sequences, preventing their further processing.
  • the Gl phase is the time at which cells commit to a new round of division in response to external and internal sequences and, thus, transcription factors which regulate express ion of Gl cyclins are surely important in controlling cell proliferation. Modulation of the transcription factors is one route by which D-type cyclin activity can be influenced, resulting, in the case of inhibition or prevention of function of the transcription factor (s), in reduced D-type cyclin activity.
  • complex formation can be prevented indirectly by degrading the D- type cyclin(s) , such as by introducing a protease or substance which enhances cyclin breakdown into cells.
  • the effect is indirect in that less D-type cyclin is available than would otherwise be the case.
  • protein kinase-D type cyclin complex formation is prevented in a more direct manner by, for example, introducing into cells a drug or other agent which binds the protein kinase or the D-type cyclin or otherwise interferes with the physical association between the cyclin and the protein kinase it activates (e.g., by intercalation) or disrupts the catalytic activity of the enzyme.
  • Peptides and small organic compounds to be used for this purpose can be designed, based on analysis of the amino acid sequences of D-type cyclins, to include residues necessary for binding and to exclude residues whose presence results in activation. This can be done, for example, by systematically mapping the binding site(s) and designing molecules which recognize or otherwise associate with the site(s) necessary for activation, but do not cause activation.
  • an agent e.g., an antibody or anti-sense or other nucleic acid molecule
  • an agent e.g., an antibody or anti-sense or other nucleic acid molecule
  • agents which selectively inhibit cyclin Dl might be expected to be particularly useful, since Dl has been shown to be differentially expressed (expressed at particularly high levels in cells of neural origin) .
  • Antibodies specifically reactive with D-type cyclins of the present invention can also be produced, using known methods.
  • anti-D type cyclin antisera can be produced by injecting an appropriate host (e.g. rabbits, mice, rats, pigs) with the D-type cyclin against which anti sera is desired and withdrawing blood from the host animal after sufficient time for antibodies to have been formed.
  • Monoclonal antibodies can also be produced using known techniques. Sambrook, J. et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) .
  • the present invention also includes a method of screening compounds or molecules for their ability to inhibit or suppress the function of a cyclin, particularly a D-type cyclin.
  • mutant cells as described herein, in which a D-type cyclin such as Dl or D3 , is expressed can be used.
  • a compound or molecule to be assessed for its ability to inhibit a D-type cyclin is contacted with the cells, under conditions appropriate for entry of the compound or molecule into the cells. Inhibition of the cyclin will result in arrest of the cells or a reduced rate of cell division. Comparison of Othe rate or extent of cell division in the presence of the compound or molecule being assessed with cell division of an appropriate control (e.g.
  • a human glioblastoma cDNA library carried in the yeast expression vector pADNS (Colicelli, J. et al . , Pro. Natl . Acad. Sci. USA 85:3599 (1989)) was introduced into the yeast .
  • the vector pADNS has the LEU2 marker, the 2 ⁇ replication origin, and the promoter and terminator sequences from the yeast alcohol dehydrogenase gene (Figure 1) . Approximately 3 x 10 6 transformants were screened for the ability to grow on glucose containing medium. After 12 days of incubation, twelve colonies were obtained. The majority of these proved to be revertants. However, in two cases, the ability to grow on glucose correlated with the maintenance of the LEU2 marker as assessed by plasmid stability tests.
  • yeast transformants carried plasmids designated pCYCDl-21 and pCYCDl-19 (see below) . Both were recovered in E. coli. Upon reintroduction into yeast, the plasmids rescued the CLN deficient strain, although the rescue was inefficient and the rescued strain grew relatively poorly.
  • the estimated molecular weight of the predicted protein product of the gene is 33,670 daltons starting from the first in- frame AUG codon at nucleotide 145 ( Figure 2) .
  • the predicted protein is related to other cyclins (see below) and has an unusually low pi of 4.9 (compared to 6.4 of human cyclin A, 7.7 of human cyclin B and 5.6 of CLNl) , largely contributed by the high concentration of acidic residues at its C- terminus.
  • CYCDl encodes the smallest (34 kd) cyclin protein identified so far, compared to the 49 kd human cyclin A, 50 kd human cyclin B and 62 kd S. cerevisiae CLNl.
  • a and B type cyclins the difference is due to the lack of almost the entire N-terminal segment that contains the so called "destruction box" identified in both A and B type cyclins (Glotzer M. et al. , Nature 349 : 132 (1991)) .
  • both 5' and 3' end sub-fragments of the XCYCDl-H12 clone were used to screen both cDNA and genomic libraries, to test whether there might be alternative transcription initiation, polyadenylation and/or mRNA splicing.
  • Two longer cDNA clones, XCYCD1-H034 (1.7 kb) from HeLa cells and XDYDC1-T078 (4.1 kb) from human teratocarcinoma cells, as well as several genomic clones were isolated and partially sequenced.
  • Both XCYCD1-H034 and XCYCD1-T078 have identical sequences to XCYCD1-H12 clone from their 5' ends ( Figure 6) . Both differ from XCYCD1-H12 in having additional sequences at the 3' end, after the site of polyadenylation. These 3' sequences are the same in XCYCD1-H034 and XCYCD1-T078, but extend further in the latter clone ( Figure 6) . Nucleotide sequencing of a genomic clone within this region revealed colinearity between the cDNAs and the genomic DNA ( Figure 6) . There is a single base deletion (an A residue) in XCYCD1-T078 cDNA clone.
  • the level of transcript is 7 to 10 fold higher in the glioblastoma cells, compared to HeLa cells. In both HeLa and U118 MG cells, both high and low molecular weight transcripts are observed.
  • the parental strain was BF305-15d (MATa leu2-3 leu2-112 his3-ll his3-15 ura3-52 trpl adel metl4 arg5,6) (Futcher, B. , et al . , Mol. Cell. Biol. 5:2213 (1986) ) .
  • the strain was converted into a conditional cln- strain in three steps. First, the chromosomal CLN3 gene was placed under control of the GAL1 promoter.
  • the ligation of the Xhol end to the EcoRI end was accomplished by filling in the ends with Klenow, and blunt-end ligating (destroying the EcoRI site) .
  • the GALl promoter had replaced the DNA normally found between -110 and -411 upstream of CLN3.
  • an EcoRI to SphI fragment was excised from this new pBF30 derivative. This fragment had extensive 5' and 3' homology to the CLN3 region, but contained the GALl promoter and a URA3 marker just upstream of CLN3.
  • Strain BF305-15d was transformed with this fragment and Ura- transformants were selected. These were checked by Southern analysis. In addition, average cell size was measured when the GALl promoter was induced or uninduced.
  • CLNl gene was disrupted.
  • a fragment of CLNl was obtained from I. Fitch, and used to obtain a full length clone of CLNl by hybridization, and this was subcloned into a pUC plasmid.
  • a BamHI fragment carrying the HIS3 gene was inserted into an Ncol site in the CLNl open reading frame.
  • a large EcoRI fragment with extensive 5' and 3' homology to the CLNl region was then excised, and used to transform the BF305-15d GAL-CLN3 strain described above. Transformation was done on YNB-his raffinose galactose plates. His+ clones were selected, and checked by Southern analysis.
  • the CLN2 gene was disrupted.
  • a fragment of CLN2 was obtained from I. Fitch, and used to obtain a full length clone of CLN2 by hybridization, and this was subcloned into a pUC plasmid.
  • An EcoRI fragment carrying the TRP1 gene was inserted into an Spel site in the CLN2 open reading frame.
  • a BamHI-Kpnl fragment was excised and used to transform the BF305-15d GAL-CLN3 HIS3: :clnl strain described above. Transformation was done on YNB-trp raffinose galactose plates. Trp+ clones were selected.
  • TRP1 fragment included an ARS
  • many of the transformants contained autonomously replicating plasmid rather than a disrupted CLN2 gene.
  • several percent of the transformants were simple TRPl::cln2 disruptants, as shown by phenotypic and Southern analysis .
  • 305- 15d #21 One particular 305-15d GAL1-CLN3 HIS3::clnl TRPl::cln2 transformant called clone #21 (referred to hereafter as 305- 15d #21) was analyzed extensively. When grown in 1% raffinose and 1% galactose, it had a doubling time indistinguishable from the CLN wild-type parental strain. However, it displayed a moderate Wee phenotype (small cell volume) , as expected for a CLN3 overexpressor. When glucose was added, or when galactose was removed, cells accumulated in Gl phase, and cell division ceased, though cells continued to increase in mass and volume. After overnight incubation in the Gl-arrested state, essentially no budded cells were seen, and a large proportion of the cells had lysed due to their uncontrolled increase in size.
  • HeLa and 293 cells were cultured at 37°C either on plates or in suspension in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum.
  • DMEM Dulbecco's modified Eagle's medium
  • Glioblastoma U118 MG cells were cultured on plates in DMEM supplemented with 15% fetal bovine serum and 0.1 mM non-essential amino acid (GIBCO) .
  • Human HeLa cell cDNA library in XZAP II was purchased from Stratagene. Human T cell cDNA library in XgtlO was a gift of M. Gillman (Cold Spring Harbor Laboratory) . Human glioblastoma U118 MG and glioblastoma SW1088 cell cDNA libraries in XZAP II were gifts of M. Wigler (Cold Spring Harbor Laboratory) . Human teratocarcinoma cell cDNA library XgtlO was a gift of Skowronski (Cold Spring Harbor Laboratory) . Normal human liver genomic library XGEM-11 was purchased from Promega.
  • RNA from cell culture was extracted exactly according to Sambrook, et al. (Sambrook, J. et al. , Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) ) using guanidium thiocyanate followed by centrifugation in CsCl solution.
  • Poly(A) +RNA was isolated from total RNA preparation using Poly (A) +Quick push columns (Stratagene) . RNA samples were separated on a 1% agarose-formaldehyde MOPs gel and transferred to a nitrocellulose filter.
  • Northern hybridizations (as well as library screening) were carried out at 68°C in a solution containing 5 x Denhardt's solution, 2 x SSC, 0.1% SDS, 100 ⁇ g/ml denatured Salmon sperm DNA, 25 ⁇ M NaP0 4 (pH7.0) and 10% dextran sulfate. Probes were labelled by the random priming labelling method (Feinberg, A. et al. , Anal . Biochem. 132:6 (1983)) .
  • a 1.3 kb Hind III fragment of cDNA clone pCYCDlH12 was used as coding region probe for Northern hybridization and genomic library screening, a 1.7 kb Hind III-EcoRI fragment from cDNA clone pCYCDl-T078 was used as 3' fragment probe.
  • Bacterial culture was lysed by sonication in a lysis buffer (5 mM EDTA, 10% glycerol, 50 mM Tris-HCL, pH 8.0, 0.005% Triton X- 100) containing 6 M urea (CYCDl encoded p34 is only partial soluble in 8 M urea) , centrifuged for 15 minutes at 20,000 g force. The pellet was washed once in the lysis buffer with 6 M urea, pelleted again, resuspended in lysis buffer containing 8 urea, and centrifuged. The supernatant which enriched the 34 kd CYCDl protein was loaded on a 10% polyacrymide gel. The 34 kd band was cut from the gel and eluted with PBS containing 0.1% SDS. Se ⁇ uence Alignment and Formation of an Evolutionary Tree
  • Protein sequence alignment was conducted virtually by eye according to the methods described and discussed in detail by Xiong and Eickbush (Xiong, Y. et al . , EMBO J. 9:3353 (1990)) . Numbers within certain sequences indicate the number of amino acid residues omitted from the sequence as the result of insertion.
  • Numbers within certain sequences indicate the number of amino acid residues omitted from the sequence as the result of insertion (e.g., for CLNl, ...TWG25RLS...- indicates that 25 amino acids have been omitted between G and R) .
  • Sources for each sequence used in this alignment and in the construction of an evolutionary tree are as follows: CYCA-Hs, human A type cyclin (Wang, J. et al . , Nature 343:555 (1990)) ; CYCA-X1, Xenopus A-type cyclin
  • CYCBl-Hs human Bl-type cyclin (Pines, J. et al., Cell 58:833 (1989) ; CYCB1-X1 and CYCB2-X1, Xenopus Bl ⁇ and B2-type cyclin (Minshull, J. et al . , Cell 55:947-956 (1989)) ; CYCB-Ss, clam B-type cyclin (Westendorf, J.M et al., J Cell Biol. 108:1431 (1989)) ; CYCB-Asp, starfish B- type cyclin (Tachibana, K. et al . , Dev. Biol.
  • lysis buffer 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM EDTA, 0.5% NP-40, 0.5% Nadeoxycholate, 1 mM PMSF
  • Immunoprecipitation was carried out using 1 mg protein from each cell lysate at 4°C for overnight.
  • 60 ⁇ l of Protein A-agarose PIERCE was added to each immunoprecipitation and incubated at 4 * C for 1 hour with constant rotating.
  • the immunoprecipitate was washed three times with the lysis buffer and final resuspended in 50 ⁇ l 2 x SDS protein sample buffer boiled for 5 minutes and loaded onto a 10% polyacrymide gel . Proteins were transferred to a nitrocellulose filter using a SDE Electroblotting System
  • the filter was blocked for 2 to 6 hours with 1 x PBS, 3% BSA and 0.1% sodium azide, washed 10 minutes each time and 6 times with NET gel buffer (50 mM Tris-HCl, pH 7.5, 150 mM
  • the tree was constructed using the Neighbor-Joining method
  • the length of horizontal line reflects the divergence.
  • the branch length between the node connecting the CLN cyclins and other cyclins was arbitrarily divided.
  • the human HeLa cell cDNA library, the human glioblastoma cell U118 MG cDNA library, the normal human liver genomic library, and the hybridization buffer were the same as those described above.
  • a human hippocampus cDNA library was purchased from Stratagene, Inc. High and low-stringency hybridizations were carried out at 68° and 50°C, respectively.
  • To prepare template DNA for PCR reactions approximately 2 million lambda phages from each cDNA library were plated at a density of 10 5 PFU/150-mm plate, and DNA was prepared from the plate lysate according to Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.
  • EXAMPLE 4 Isolation of Human Cyclin D2 and D3 cDNAs
  • the first 5' oligonucleotide primer, HCND11 is a 8 1 9 2 - f o l d d e g e n e r a t e 3 8 - m e r (TGGATG[T/C] TNGA[A/G] GTNTG[T/C] GA[A/C] GA[A/G] CA- [A/G]AA[A/ G]TG[T/C]GA[A/G]GA) (SEQ ID No. 37) , encoding 13 amino acids
  • the second 5' oligonucleotide primer, HCND12 is a 8192-fold degenerate
  • HCND13 is a 3072-fold degenerate 24- mer ( [A/G] TCNGT [A/G] TA[A/G/T]AT [A/G] CANA[A/G] [T/C] TT-
  • PCR reactions were carried out for 30 cycles at 94°C for 1 min, 48°C for 1 min, and 72°C for 1 min.
  • the reactions contained 50 mM KC1, 10 mM Tris-HCl (pH 8.3) , 1.5 mM MgCl 2 , 0.01% gelatin, 0.2 mM each of dATP, dGTP, dCTP, and dTTP, 2.5 units of Tag polymerase, 5 ⁇ M of oligonucleotide, and 2-10 ⁇ g of template DNA.
  • PCR products generated by HCNDll and HCND13 were verified in a second- round PCR reaction using HCND12 and HCND13 as the primers. After resolution on a 1.2% agarose gel, DNA fragments with the expected size (200 bp between primer HCNDll and HCND13) were purified and subcloned into the Smal site of phagmid vector pUC118 for sequencing.
  • the 201-bp fragment of the D3 PCR product was labeled with oligonucleotide primers HCNDll and HCND13 using a random-primed labeling technique (Feinberg, A. P. et al . , Anal. Biochem. 132:6
  • the probe used to screen the human genomic library for the CCND3 gene was a 2-kb EcoRI fragment derived from cDNA clone XD3-H34. All hybridizations for the screen of human cyclin D3 were carried out at high stringency.
  • PCR clones corresponding to CCNDl and CCND3 have been repeatedly isolated from both cDNA libraries; CCND2 has not.
  • cyclin D2 a 1-kb EcoRI fragment derived from mouse cy!2 cDNA was used as a probe to screen a human genomic library. Under low-stringency conditions, this probe hybridized to both human cyclins Dl and D2.
  • the cyclin Dl clones were eliminated through another hybridization with a human cyclin Dl probe at high stringency.
  • Human CCND2 genomic clones were subsequently identified by partial sequencing and by comparing the predicted protein sequence with that of human cyclins Dl and D3 as well as mouse cy!2.
  • human CCNDl (cyclin Dl) was isolated by rescuing a triple Cln deficiency mutant of Saccharomyces cerevisiae using a genetic complementation screen. Evolutionary proximity between human and mouse, and the high sequence similarity among cyll, cy!2, and cy!3, suggested the existence of two additional D-type cyclin genes in the human genome.
  • the PCR technique was first used to isolate the putative human cyclin D2 and D3 genes. Three degenerate oligonucleotide primers were derived from highly conserved regions of human CCNDl, mouse cyll, cy!2 , and cy!3.
  • mouse cy!2 cDNA was used as a heterologous probe to screen a human cDNA library at low stringency. This resulted, initially, in isolation of 10 clones from the HeLa cell cDNA library, but all corresponded to the human cyclin Dl gene on the basis of restriction mapping. Presumably, this was because cyclin D2 in HeLa cells is expressed at very low levels. Thus, the same probe was used to screen a human genomic library, based on the assumption that the representation of Dl and D2 should be approximately equal .
  • XD2-P3 The DNA sequence of XD2-P3 revealed an open reading frame that could encode a 289-amino-acid protein with a 33,045-Da calculated molecular weight.
  • human cyclin Dl there is neither methionine nor stop codons 5' to the presumptive initiating methionine codon for both XD2-P3 (nucleotide position 22, Figure 3) and XD3-H34 (nucleotide position 101, Figure 4) .
  • both XD2-P3 and XD3-H34 are believed to contain full-length coding regions.
  • chromosome localization of CCND2 and CCND3 was determined by fluorescence in situ hybridization. Chromosome in situ suppression hybridization and in situ hybridization banding were performed as described previously (Lichter, T. et al., Science 247:64 (1990) ; Baldini, A. et al . , Genomics 9:770 (1991)) . Briefly XD2-G4 and XD3-G9 lambda genomic DNAs containing inserts of 15 and 16 kb, respectively, were labeled with biotin-11-dUTP (Sigma) by nick-translation (Brigatti, D. J. et al. , Urology 125:32 (1983) ; Boyle, A.
  • Probe size ranged between 200 and 400 nucleotides, and unincorporated nucleotides were separated from probes using Sephadex G-50 spin columns (Sambrook, J. et al. , Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989) .
  • Metaphase chromosome spreads prepared by the standard technique (Lichter, T. et al . , Science 247:64 (1990) ) were hybridized in situ with biotin-labeled D2-G4 or D3-G9.
  • Denaturation and preannealing of 5 ⁇ g of DNase- treated human placental DNA, 7 ⁇ g of DNased salmon sperm DNA, and 100 ng of labeled probe were performed before the cocktail was applied to Alu prehybridized slides.
  • the in situ hybridization banding pattern used for chromosome identification and visual localization of the probe was generated by cohybridizing the spreads with 40 ng of an Alu 48-mer oligonucleotide. This Alu oligo was chemically labeled with digoxigenin-11-dUTP (Boehringer-Mannheim) and denatured before being applied to denatured chromosomes.
  • Biotin-labeled DNA was detected using fluorescence isothiocyanate (FITC) -conjugated avidin DCS (5 ⁇ g/ml) (Vector Laboratories) ; digoxigenin-labeled DNA was detected using a rhodamine-conjugated anti-digoxigenin antibody (Boehringer-Mannheim) .
  • FITC fluorescence isothiocyanate
  • avidin DCS 5 ⁇ g/ml
  • digoxigenin-labeled DNA was detected using a rhodamine-conjugated anti-digoxigenin antibody (Boehringer-Mannheim) .
  • Fluorescence signals were imaged separately using a Zeiss Axioskop-20 epifluorescence microscope equipped with a cooled CCD camera (Photometries CH220) . Camera control and image acquisition were performed using an Apple Macintosh IIX computer. The gray scale images were pseudocolored and merged electronically as described previously (Baldini, A. et al . , Genomics 9:770
  • the Alu 48-mer R-bands consistent with the conventional R- banding pattern, were imaged and merged with images generated from the D2-G4 and D3-G9 hybridized probes.
  • the loci of D2-G4 and D3-G9 were visualized against the Alu banding by merging the corresponding FITC and rhodamine images.
  • This merged image allows the direct visualization of D2-G4 and D3-G9 on chromosomes 12 and 6, respectively.
  • the D2-G4 probe lies on the positive R-band 12pl3, while D3- G9 lies on the positive R-band 6p21.
  • Genomic clones of human D-type cyclins were isolated and characterized to study the genomic structure and to obtain probes for chromosomal mapping.
  • the entire 1.3-kb cyclin Dl cDNA clone was used as probe to screen a normal human liver genomic library.
  • Five million lambda clones were screened, and three positives were obtained.
  • lambda clone G6 was chosen for further analysis.
  • a 1.7-kb BamHI restriction fragment of XD1-G6 was subcloned into pUC118 and completely sequenced. Comparison with the cDNA clones previously isolated and RNase protection experiment results (Withers, D.A. et al., Mol. Cell. Biol.
  • the 8 lambda clones that did not hybridize with the human Dl probe at high stringency fall into three classes represented by XD2-G1, XD2-G2, and XD2-G4, respectively. These three lambda clones were subcloned into a pUC plasmid vector, and small restriction fragments containing coding region were identified by Southern hybridization using a mouse cy!2 cDNA probe. A 0.4-kb BamHI fragment derived from XD2-G1 was subsequently used as a probe to screen a human hippocampus cell cDNA library at high stringency.
  • the 2.7-kb Sacl-Smal fragment contains 1620 bp of sequence 5' to the presumptive initiating methionine codon identified in D2 cDNA ( Figure 3) and a 195- bp exon followed by a 907-bp intervening sequence.
  • Lambda genomic clones corresponding to the human cyclin D3 were isolated from the same genomic library using human D3 cDNA as a probe. Of four million clones screened, nine were positives.
  • a 2.0-kb Hindlll-Scal restriction fragment from XD3-G5 and a 3.7-kb Sacl-Hindlll restriction fragment from XD3-G9 were further subcloned into a pUC plasmid vector for more detailed restriction mapping and complete sequencing, as they both hybridized to the 5' cyclin D3 cDNA probe.
  • the 3.7-kb fragment from clone G9 contains 1.8 kb of sequence 5' to the presumptive initiating methionine codon identified in D3 cDNA ( Figure 4) , a 198-bp exon 1, a 684-bp exon 2, and a 870-bp intron.
  • the 1.5-kb Bcll-Bglll fragment subcloned from clone XD2-G1 has been completely sequenced and compared with cyclin D2 cDNA clone XD2-P3. As shown in Figure 10, it contains three internal stop codons (nucleotide positions 495, 956, and 1310, indicated by asterisks) , two frameshifts (position 1188 and 1291, slash lines) , one insertion, and one deletion. It has also accumulated many missense nucleotide substitutions, some of which occurred at the positions that are conserved in all cyclins.
  • triplet CGT at position 277 to 279 of D2 cDNA encodes amino acid Arg, which is an invariant residue in all cyclins (see Figure 8) .
  • a nucleotide change from C to T at the corresponding position (nucleotide 731) in clone XD2-G1 ( Figure 10) gave rise to a triplet TGT encoding Cys instead of Arg.
  • Sequencing of the 2.0-kb Hindlll-Scal fragment from clone XD3-G5 revealed a cyclin D3 pseudogene ( Figure 11) .
  • nucleotide position 1265 In addition to a nonsense mutation (nucleotide position 1265) , two frameshifts (position 1210 and 1679) , a 15-bp internal duplication (underlined region from position 1361 to 1376) , and many missense mutations, a nucleotide change from A to G at position 1182 resulted in an amino acid change from the presumptive initiating methionine codon ATG to GTG encoding Val .
  • clones XD2-G1 and XD3-G5 contain pseudogenes of cyclins D2 and D3 , respectively.
  • MOLECULE TYPE DNA (genomic)
  • Val Lys Phe lie Ser Asn Pro Pro Ser Met Val Ala Ala Gly Ser Val 195 200 205
  • MOLECULE TYPE DNA (genomic)
  • CTGTCTCTGA TCCGCAAGCA TGCTCAGACC TTCATTGCTC TGTGTGCCAC CGACTTTAAG 660
  • TTTGCCATGT ACCCACCGTC GATGATCGCA ACTGGAAGTG TGGGAGCAGC CATCTGTGGG 720
  • Lys Asp lie Gin Pro Tyr Met Arg Arg Met Val Ala Thr Trp Met Leu 50 55 60
  • Lys Glu Thr Ser Pro Leu Thr Ala Glu Lys Leu Cys lie Tyr Thr Asp 115 120 125
  • Glu His lie Leu Arg Lys Leu Pro Gin Gin Gin Arg Glu Lys Leu Ser Leu 165 170 175 lie Arg Lys His Ala Gin Thr Phe lie Ala Leu Cys Ala Thr Asp Phe 180 185 190
  • Ala Ala lie Cys Gly Leu Gin Gin Asp Glu Glu Val Ser Ser Leu Thr 210 215 220
  • MOLECULE TYPE DNA (genomic)
  • Gly Lys Leu Lys Trp Asp Leu Ala Ala Val lie Ala His Asp Phe Leu 145 150 155 160
  • Gin Met Asp lie Leu Glu Tyr Phe Arg Glu Ser Glu Lys Lys His Arg 225 230 235 240
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • GCCCACTGCC CAATCCTCAC CTCTCTTCTC CTCCACCTTC TGTCTCTGCC CTCACCTCTC 240 CTCTGAAAAC CCCCTATTGA GCCAAAGGAA GGAGATGAGG GGAATGCTTT TGCCTTCCCC 300
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • GCAGCCCCCT CACGCTCACG AATTCAGTCC CAGGGCAAAT TCTAAAGGTG AAGGGACGTC 120
  • MOLECULE TYPE DNA (genomic)
  • GTCTCTCCCC TTCCTCCTGG AGTGAAATAC ACCAAAGGGC GCGGTGGGGG TGGGGGGTGA 120
  • TTGTCAGCAG ATGCAGGGGC GAGGAAGCGG GTTTTTCCTG CGTGGCCGCT GGCGCGGGGG 540
  • CTCCAGAGAA GCACCCCCCT TCCTTCCTAA TACCCACCTC TCCCTCCCTC TTCTTCCTCT 720 GCACACACTC TGCAGGGGGG GGCAGAAGGG ACGTTGTTCT GGTCCCTTTA ATCGGGGCTT 780
  • MOLECULE TYPE DNA (genomic)
  • GCAGCCCCCT CACGCTCACG AATTCAGTCC CAGGGCAAAT TCTAAAGGTG AAGGGACGTC 120
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention se rapporte à une nouvelle classe de cyclines, appelées cyclines de type D, d'origine mammifère et plus particulièrement d'origine humaine. L'invention décrit également: des ADN et des ARN codants pour ces nouvelles cyclines; un procédé d'identification d'autres cyclines de type D et de type non D; un procédé pour détecter un niveau accru de cycline de type D et un procédé pour inhiber la division cellulaire en perturbant la formation du complexe protéine-kinase/cycline de type D, qui est essentiel pour le démarrage du cycle cellulaire.
PCT/US1993/005000 1992-05-26 1993-05-25 Cycline de type d et utilisations correspondantes WO1993024514A1 (fr)

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ONCOGENE, Volume 6, No. 3, issued March 1991, ROSENBERG et al., "Rearrangement and Overexpression of D11S287E, a Candidate Oncogene on Chromosome 11q13 in Benign Parathyroid Tumors", p. 449-453. *

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US6075123A (en) * 1996-06-03 2000-06-13 St. Jude Children's Research Hospital Cyclin-C variants, and diagnostic and therapeutic uses thereof
US6306648B1 (en) 1996-06-03 2001-10-23 St. Jude Children's Research Hospital Cyclin-C variants, and diagnostic and therapeutic uses thereof
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US7455964B1 (en) 1996-07-15 2008-11-25 The Hospital For Sick Children Genes from the 20q13 amplicon and their uses
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US8101370B2 (en) 1996-07-15 2012-01-24 The Regents Of The University Of California Genes from the 20q13 amplicon and their uses
WO1998002539A1 (fr) * 1996-07-15 1998-01-22 The Regents Of The University Of California GENES ISSUS DE L'AMPLICON 20q13 ET LEURS UTILISATIONS
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US6387919B1 (en) 1997-09-05 2002-05-14 Glaxo Wellcome Inc. Substituted oxindole derivatives as protein tyrosine kinase and as protein serine/threonine kinase inhibitors
US6541503B2 (en) 1997-09-05 2003-04-01 Smithkline Beecham Corporation Substituted oxindole derivatives as protein tyrosine kinase and as protein serine/threonine kinase inhibitors
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US6369086B1 (en) 1997-09-05 2002-04-09 Smithkline Beecham Corporation Substituted oxidole derivatives as protein tyrosine and as protein serine/threonine kinase inhibitors
WO1999022002A1 (fr) * 1997-10-24 1999-05-06 Cropdesign N.V. Nouveau cycline mitogene et son utilisation
US6624171B1 (en) 1999-03-04 2003-09-23 Smithkline Beecham Corporation Substituted aza-oxindole derivatives
US6818632B2 (en) 1999-03-04 2004-11-16 Smithkline Beecham Corporation 3-(anilinomethylene)oxindoles
US6815439B2 (en) 1999-03-04 2004-11-09 Smithkline Beecham Corporation Substituted aza-oxindole derivatives
US7129253B2 (en) 1999-03-04 2006-10-31 Smithkline Beecham Corporation Compounds
US6492398B1 (en) 1999-03-04 2002-12-10 Smithkline Beechman Corporation Thiazoloindolinone compounds
US6350747B1 (en) 1999-03-04 2002-02-26 Glaxo Wellcome Inc. 3-(anilinomethylene) oxindoles
US6620818B1 (en) 2000-03-01 2003-09-16 Smithkline Beecham Corporation Method for reducing the severity of side effects of chemotherapy and/or radiation therapy
WO2003012059A3 (fr) * 2001-08-01 2003-12-04 Isis Pharmaceuticals Inc Modulation antisens de l'expression de la cycline d2
WO2003012059A2 (fr) * 2001-08-01 2003-02-13 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de la cycline d2
US6492173B1 (en) * 2001-08-01 2002-12-10 Isis Pharmaceuticals, Inc. Antisense inhibition of cyclin D2 expression

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