Catalytic domain of the human effector cell cycle checkpoint...

Details Catalytic domain of the human effector cell cycle checkpoint... Catalytic domain of the human effector cell cycle checkpoint...

1999-12-14

2003-12-30

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S235100, C435S348000, C435S254110, C435S252300, C435S419000, C435S325000, C435S320100, C536S023200, C536S023500

Reexamination Certificate

active

06670167

ABSTRACT:

FIELD OF THE INVENTION
The present invention generally relates to cell cycle checkpoint kinases which are essential to cellular DNA damage responses and coordinating cell cycle arrest. The checkpoint kinases play a role in the surveillance and response to DNA damage. The damage may result from external or internal forces. Such forces include but are not limited to errors in replication, DNA base damage, DNA strand breaks, or exposure to radiation or cytotoxic chemicals. These checkpoint kinases are integral in the regulatory pathways leading to cell cycle arrest and apoptosis following DNA damage, giving the cell notice and time to correct lesions prior to the initiation of replication and chromosome separation. The present invention more specifically relates to the isolation and purification of the catalytic domain of the human effector checkpoint protein kinase (hChk1) and its use in the discovery, identification and characterization of inhibitors of same.
BACKGROUND
Cell growth, division and death is essential to the life cycle of multi-celled organisms. These processes and their regulation are strikingly similar across all eukaryotic species. Somatic cell division consists of two sequential processes: DNA replication followed by chromosomal separation. The cell spends most of its time preparing for these events in a growth cycle (interphase) which in turn consists of three subphases: initial gap (G
1
), synthesis (S), and secondary gap (G
2
). In G
1
, the cell, whose biosynthetic pathways were slowed during mitosis, resumes a high rate of biosynthesis. The S phase begins when DNA synthesis starts and ends when the DNA content of the nucleus has doubled. The cell then enters G
2
, which lasts until the cell enters the final phase of division, mitotic (M). The M phase begins with nuclear envelope breakdown, chromosome condensation and formation of two identical sets of chromosomes which are separated into two new nuclei. This is followed by cell division (cytokinesis) in which each nuclei is separated into two daughter cells, which terminates the M phase and marks the beginning of interphase for the new cells.
The sequence in which the cell cycle events proceed is tightly regulated such that the initiation of one cell cycle event is dependent upon the successful completion of the prior cell cycle event. The process of monitoring genome integrity and preventing cell cycle progress in the event of DNA damage has been described as a ‘cell cycle checkpoint’ (Hartwell, L H et al.,
Science
, 246:629-634 (1989); Weinert et al.,
Genes and Dev
., 8:652 (1994)]. Cell cycle checkpoints consist of signal transduction cascades which couple DNA damage detection to cell cycle progression. Checkpoints are control systems that coordinate cell cycle progression by influencing the formation, activation and subsequent inactivation of the cyclin-dependent kinases. Checkpoint enzymes are responsible for maintaining the order and fidelity of events of the cell cycle by blocking mitosis in response to unreplicated or damaged DNA. These enzymes prevent cell cycle progression at inappropriate times, maintain the metabolic balance of cells while the cell is arrested and in some instances can induce apoptosis (programmed cell death) when the requirements of the checkpoint have not been met (O'Connor, P M,
Cancer Surveys
, 29, 151-182 (1997); Nurse, P,
Cell
, 91, 865-867 (1997); Hartwell, L H et al,
Science
, 266, 1821-1828 (1994); Hartwell, L H et al.,
Science
, 246, (1989), supra).
One series of checkpoints monitors the integrity of the genome. Upon sensing DNA damage, these “DNA damage checkpoints” block cell cycle progression in G
1
& G
2
phases, and slow progression through S phase (O'Connor, P M,
Cancer Surveys
, 29 (1997), supra; Hartwell, L H et al,
Science
, 266, (1994), supra). This action enables DNA repair to be completed before replication of the genome and subsequent separation of this genetic material into new daughter cell takes place.
Various mutations associated with malignancy affect the cancer cells ability to regulate checkpoints, allowing cells with DNA damage the increased likelihood to continue replicating and to escape damage-mediated apoptosis These factors contribute to the genomic instability which drives the genetic evolution of human cancers and contributes to the resistance of cancer cells to most current chemotherapy and radiotherapy intervention.
Due to abnormalities in the p53 tumor suppressor pathway, most cancer cells lack a functional G
1
checkpoint control system. This makes them particularly vulnerable to abrogation of the last remaining barrier protecting them from the cancer killing effects of DNA damaging agents: the G
2
checkpoint. The G
2
DNA damage checkpoint ensures maintenance of cell viability by delaying progression into mitosis in cells that have suffered genomic damage. The G
2
checkpoint is controlled by cell cycle checkpoint pathways which inhibit mitosis if previous events are incomplete or if the DNA is damaged. This regulation control system has been conserved from yeast to humans. Important in this conserved system is a kinase, Chk1 (or p56Chk1), which transduces signals from the DNA damage sensory complex to inhibit activation of the cyclin B/Cdc2 kinase which promotes mitotic entry (Peng, C Y et al,
Science
, 277, 1501-1505 (1997); Sanchez Y, et al.,
Science
, 277, 1497-1501 (1997); Walworth, N et al.,
Nature
, 363(6427), 368-71 (May 27, 1993); al-Khodairy et al.,
Mol Biol Cell
, 5(2):147-60 (Febuary 1994); Carr et al.,
Curr Biol
., 5(10): 1179-90 (Oct. 1, 1995)). The repair checkpoint kinase, Chk1, regulates Cdc25, a phosphatase that activates Cdc2. Thus, Chk1 serves as the direct link between the G
2
checkpoint and the negative regulation of Cdc2.
Inactivation of Chk1 has been shown to both abrogate G
2
arrest induced by DNA damage inflicted by either anticancer agents or endogenous DNA damage, as well as, result in preferential killing of the resulting checkpoint defective cells (Nurse, P,
Cell
, 91, (1997), supra; Weinert, T,
Science
, 277, 1450-1451 (1997); Walworth, N et al.,
Nature
, 363, (1993) supra; al-Khodairy et al.,
Molec. Biol. Cell
, 5, (1994), supra; Wan, S et al.,
Yeast
, 15(10A), 821-8 (July 1999)).
The fact that cancer cells have also been shown to be more vulnerable to G
2
checkpoint abrogation has encouraged the pursuit of G
2
checkpoint abrogating drugs (Wang, Q et al.,
PNAS
96: 3706-3711 (1999); Fan, S et al.,
Cancer Res
., 55, 1649-1654 (1995); Powell, S N et al.,
Cancer Res
., 55, 1643-1648 (1995); Russell, K J et al.,
Cancer Res
., 55, 1639-1642 (1995); Wang, Q et al.,
J Natl Cancer Inst
., 88, 956-967 (1996)). Such checkpoint abrogating drugs could improve the killing of tumors exposed to DNA damaging events including that inflicted by therapeutic agents, hypoxic-stress induced because of a limited blood supply (anti-angiogenic agents), or endogenous DNA damage arising as a consequence of a cancer cell's inherent genomic instability. Selective manipulation of checkpoint control in cancer cells can afford broad utilization in cancer chemotherapeutic and radiotherapy regimens and may in addition, offer a common hallmark of human cancer “genomic instability” to be exploited as the selective basis for the destruction cancer cells.
A number of lines of evidence place Chk1 as a pivotal target in DNA damage checkpoint control. However, Chk1 is a difficult enzyme to study because the full length protein is not the most active form of Chk1. While others have examined the nucleotide and amino acid sequence of the full-length checkpoint kinase and estimated the location of the kinase domain, there is a need for the isolation and purification of the kinase domain of Chk1and the maintenance of its catalytically active conformation.
SUMMARY OF THE INVENTION
The generation, kinetic characterization, and structure determination of the kinase domain of the human Chk1 protein is disclosed herein. The domain begins between residues 1 and 16 and terminates between residues 265 and 29

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