Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-08-10
2001-04-03
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C435S358000
Reexamination Certificate
active
06210924
ABSTRACT:
BACKGROUND OF THE INVENTION
Progression through the mammalian cell cycle is driven by orderly activation of cyclin-dependent kinases (CDKs). An active CDK is composed of a catalytic subunit and a regulatory subunit called cyclin. CDK activity is regulated through interactions with cyclins and CDK inhibitors (CKIs) and by post-translational modifications (e.g., phosphorylation).
Transition between cell cycle states is regulated at defined checkpoints by different cyclin subunits: G1 cyclins for the G1/S transition, S cyclins for progression through S phase, and G2 or mitotic cyclins for entry into mitosis. The commitment of a cell to enter the S phase occurs at a restriction point (R) late in G1, after which mitogenic growth factors are no longer required for cells to complete division.
Cyclins D and E are synthesized sequentially during G1 and are rate-limiting for S-phase entry, so they can be viewed as G1 cyclins. At least three mammalian genes encode D-type cyclins (D1, D2 and D3). D-type cyclins are progressively induced as part of the delayed early response to mitogenic stimulation, and they are expressed in a cell lineage-specific fashion. Assembly of D-type cyclins with CDK4 and CDK6 is regulated post-translationally by mitogens. Once assembled, cyclin D-bound CDKs must be phosphorylated by a CDK-activating kinase (CAK) to acquire catalytic activity.
Cyclin D genes are on a different branch of the evolutionary tree from A-, B-, or E-type cyclin, and D-type cyclins have some different properties from other cyclins. They are short-lived proteins (t
½
<25 min). Withdrawal of growth factors during G1 prevents steady accumulation of cyclin D, correlating with the failure of growth factor-deprived cells to progress past the R point. Thus, expression of cyclin D is regulated by extracellular signals, unlike the periodic expression of cyclins A, B and E.
Overexpression of human cyclins D1 and E in rodent or human fibroblasts shortens G1, decreases cell size, and reduces the serum requirement for the G1-to-S transition (Resnitzky et al,
MCB ,
4 1669-79 (1994); Quelle etal.,
Genes & Development
7, 1559-71 (1993); Ohtsubo & Roberts,
Science
259, 1908-12 (1992)). These results suggest that D cyclins might override a function physiologically regulated by cyclin E or vice-versa. However, overexpression of cyclin D or E does not lead to fibroblast transformation—cells remain serum-dependent, contact-inhibited, and unable to form colonies in semisolid medium. Overexpression of cyclin D1 has also been found to enhance endogenous gene amplification, suggesting that it plays a role in genomic instability during tumor development. Zhou et al.,
Cancer Res.
56: 36-9 (1996).
SUMMARY OF THE INVENTION
The present invention concerns a eukaryotic cell comprising:
(a) a cyclin D gene product, wherein the cyclin D gene product is functionally expressed in the cell at a level greater than any native level of expression; and
(b) a protein of interest, wherein the protein of interest is expressed in the cell at a level greater than any native level of expression.
The present invention also concerns a process for producing a protein of interest, which comprises:
A. creating a eukaryotic cell that expresses
1. a cyclin D gene product at a level greater than any native level of expression and
2. a protein of interest at a level greater than any native level of expression;
B. culturing the cell under conditions permitting expression of the protein of interest and functional expression of the cyclin D gene product; and
C. isolating the protein of interest.
The cells of this invention are preferably mammalian, with CHO cells most preferred. The cyclin D gene product is preferably mammalian, with human origin most preferred. The cyclin D gene or the gene encoding the protein of interest (or both) may be comprised in an expression vector or vectors inside the cell or may be integrated into the cell genome.
Any number of proteins of interest may be used in the present invention. Specifically, EPO, OPG, leptin and NESP, and any derivatives thereof may be employed.
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Diamond et al. (1981), “Monoclonal Antibodies: A New Technology for Producing Serologic Reagents”The New England Journal of Medicine304:1344-1349.
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Quelle et al, (1993), “Overexpression of mouse D-type cyclins accelerates G1phase in rodent fibroblasts”Genes&Development7:1559-1571.
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Zieg et al., Properties of single-step mutants of syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, Mol. Cell. Biol. 3: 2089-2098, 1983.*
Ewen et a l., Cell 73: 487-497, Functional interactions of the retinoblastoma protein with mammalian D-type cyclins, May 1993.*
Hiari and Sherr, Mol. Cell. Biol. 16:6457-6467, Interaaction of D-type cyclins with a novel myb-like transcription factor, DMP1, Nov. 1996.
Brankow David William
Gudas Jean Marie
Hu Shaw-Fen Sylvia
Amgen Inc.
Davis Katharine F
Gaul Timothy J.
Levy Ron K.
Odre Steven M.
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