Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1998-02-20
2001-04-17
Nolan, Patrick J. (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387100, C530S389200
Reexamination Certificate
active
06218515
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to cyclin E variants, and more particularly to constitutively active truncated human cyclin E proteins and uses thereof.
BACKGROUND OF THE INVENTION
Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
Cyclins are prime cell cycle regulators and central to the control of cell proliferation in eukaryotic cells via their association with and activation of cyclin-dependent protein kinases 1-7 (cdks) (reviewed in, Elledge et al., 1991; Heichman et al., 1994; Hunter et al., 1994; King et al., 1994; Morgan, 1995; Nurse, 1994; Sherr, 1994). Cyclins were first identified in marine invertebrates as a result of their dramatic cell cycle expression patterns during meiotic and early mitotic divisions (Evans et al., 1983; Sherr, 1993; Standart et al., 1987; Swenson et al., 1986). Several classes of cyclins have been described and are currently designated as cyclins A-H, some with multiple members (reviewed in Draetta, 1994). Cyclins can be distinguished on the basis of conserved sequence motifs, patterns of appearance and apparent functional roles during specific phases and regulatory points of the cell cycle in a variety of species.
The connection between cyclins and cancer has been substantiated with the D type cyclins (Draetta, 1994; Hunter et al., 1991; Hunter & Pines, 1994; Sherr, 1993). Cyclin D1 was identified simultaneously by several laboratories using independent systems: It was identified in mouse macrophages due to its induction by colony stimulating factor 1 during G1 (Matsushime et al., 1991); in complementation studies using yeast strains deficient in G1 cyclins (Lew et al., 1991; Xiong et al., 1991); as the product of the bcl-1 oncogene (Withers et al., 1991), and as the PRAD1 proto-oncogene in some parathyroid tumors where its locus is overexpressed as a result of a chromosomal rearrangement that translocates it to the enhancer of the parathyroid hormone gene (Matsushime, et al., 1991; Motokura et al., 1993; Motokura et al., 1991; Quelle et al., 1993). In centrocytic B cell lymphomas cyclin D1 (PRAD1)/BCL1 is targeted by chromosomal translocations at the BCL1 breakpoint, t(11;14)(q13;q32) (Rosenberg et al., 1991a; Rosenberg et al., 1991b). Furthermore, the cyclin D1 locus undergoes gene amplification in mouse skin carcinogenesis, as well as in breast, esophageal, colorectal and squamous cell carcinomas (Bianchi et al., 1993; Buckley et al., 1993; Jiang et al., 1992; Jiang et al., 1993b; Lammie et al., 1991; Leach et al., 1993). Several groups have examined the ability of cyclin D1 to transform cells directly in culture with mixed results (Hinds et al., 1994; Hinds et al., 1992; Jiang et al., 1993a; Lovec et al., 1994; Musgrove et al., 1994; Quelle, et al., 1993; Resnitzky et al., 1994; Rosenwald et al., 1993; Sherr, 1993). However, the overexpression of cyclin D1 was recently observed in mammary cells of transgenic mice and results in abnormal proliferation of these cells and the development of mammary adenocarcinomas (Wang et al., 1994). This observation strengthens the hypothesis that the inappropriate expression of a G1 type cyclin may lead to loss of growth control.
Recently, the linkage between oncogenesis and the cell cycle has been reinforced by correlating the deranged expression of cyclins to the loss of growth control in breast cancer (Buckley, et al., 1993; Keyomarsi et al., 1993). Using proliferating normal versus human tumor breast cell lines in culture as a model system, several changes that are seen in all or most of these lines have been described. These include increased cyclin mRNA stability, resulting in overexpression of mitotic cyclins and cdk2 RNAs and proteins in 9/10 tumor lines, leading to the deranged order of appearance of mitotic cyclins prior to Gi cyclins in synchronized tumor cells.
The most striking abnormality in cyclin expression found was that of cyclin E. Cyclin E protein not only was overexpressed in 10/10 breast tumor cell lines but it was also present in lower molecular weight isoforms than that found in normal cells (Keyomarsi & Pardee, 1993). One possibility for the presence of multiple transcripts of cyclin E is due to alternative splicing. Precedent for alternative splicing of cyclin E has recently been reported by Ohtsubo et. al. where they identified a longer form of cyclin E (cyclin E-L) which contains 15 amino acids at the amino terminus which through alternative splicing, is absent in the original form of cyclin E (cyclin E wt) (Ohtsubo et al., 1995). In addition Sewing et al. also identified another splice variant of cyclin E, termed cyclin Es (Sewing et al., 1994). Cyclin Es lacks 49 amino acids within the cyclin box, and is 90% less abundant than the wild-type cyclin E sequence. This form is unable to associate with cdk2, is inactive in histone Hl kinase assays, and is unable to rescue a triple CLN mutation of S. cerevisiae (Sewing, et al., 1994). The cyclin box is a consensus region which confers activity by its association to a cdk (Lees et al., 1992). The relevance of cyclin derangement to in vivo conditions was directly examined, by measuring the expression of cyclin E protein in tumor samples versus normal adjacent tissue obtained from patients with various malignancies (Keyomarsi et al., 1994). These analyses revealed that breast cancers and other solid tumors, as well as malignant lymphocytes from patients with lymphatic leukemia, show severe quantitative and qualitative alteration in cyclin E protein expression independent of the S-phase fraction of the samples. In addition, the alteration of cyclin E becomes more severe with breast tumor stage and grade and is more consistent than cell proliferation or other tumor markers such as PCNA or c-erb B2. These observations strongly suggested the use of cyclin E as a new prognostic marker.
Therefore, a need exists to further characterize the alterations of cyclin E in breast cancer.
SUMMARY OF INVENTION
To this end, the subject invention shows that while cyclin E is cell cycle regulated in normal cells, it is present constitutively and in an active cdk2 complex in synchronized populations of breast cancer cells. Two truncated variant forms of cyclin E mRNA are detected by RT-PCR, which are ubiquitously detected in normal and tumor cells and tissues. These variant forms of cyclin E can give rise to an active cyclin/cdk2 complex in vitro, but they do not seem to be translated in normal cells.
It is an object of the subject invention to identify truncated human cyclin E protein forms which are constitutively active. It is a further object of the subject invention to provide DNA and amino acid sequences of these truncated forms of human cyclin E protein, so that the DNA sequence can be used to devise antisense nucleic acid (for use in blocking translation of mRNA encoding the truncated human cyclin E protein forms, thereby decreasing expression of the truncated human cyclin E protein forms in cells), and so that the amino acid sequence can be used to construct fragments of the truncated human cyclin E protein forms (for production of antibody to the truncated human cyclin E protein form, which can be used for detection of the truncated form).
In accordance with these objectives, the subject invention provides an isolated nucleic acid molecule encoding a truncated human cyclin E protein, the truncated human cyclin E protein being a constitutively active form of human cyclin E protein. The invention also provides an antisense nucleic acid molecule complementary to the mRNA encoding the truncated human cyclin E protein, or a fragment thereof capable of hybridizing under stringent conditions to the mRNA.
The invention further provides an isolated fragment of the nucleic acid molecule encoding the truncated human cyclin E protein. Preferably, the truncated forms of human cyclin E pr
Braman & Rogalskyj LLP
Health Research Incorporated
Nolan Patrick J.
LandOfFree
Cyclin E variants and use thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Cyclin E variants and use thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cyclin E variants and use thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2549147