Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-02-24
2004-11-23
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210, C435S007230, C435S007240, C435S007400, C436S800000
Reexamination Certificate
active
06821740
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods, reagents, and reagent kits for detecting discrete functional conformations of proteins concurrently in individual cells, particularly by flow cytometry. In particular, the invention relates to methods, reagents, and reagent kits for the determination of the phosphorylation status of the retinoblastoma susceptibility gene protein (pRB) in individual cells using multiparameter flow cytometry.
BACKGROUND OF THE INVENTION
The recent revolution in genomics technology, and in particular, the development of high density nucleic acid microarrays, has focused extraordinary attention on the differential expression of genes as markers of cellular differentiation, prognosticators of disease, and potential targets for interventional therapy. Brown et al.,
Nature Genet
. 21 (Suppl.):33-37 (1999); Duggan et al.,
Nature Genet
. 21 (Suppl.):10-14 (1999); Cole et al.,
Nature Genet
. 21 (Suppl.):38-41 (1999); Debouck et al.,
Nature Genet
. 21 (Suppl.):48-50 (1999). Although an understanding of the cell's transcriptional program is indeed important to all of these goals, the function of many critical proteins is regulated, at least in part, at the posttranslational level, a level to which transcription-based approaches are perforce indifferent.
One such critical protein is that encoded by the retinoblastoma susceptibility gene (pRB; pRb), which plays a pivotal role in the regulation of the cell cycle. pRB restrains cell cycle progression by maintaining a checkpoint in late G
1
that controls commitment of cells to enter S phase. The critical role that pRB plays in cell cycle regulation explains its status as archetypal tumor suppressor: loss of pRB function results in an inability to maintain control of the G
1
checkpoint; unchecked progression through the cell cycle is, in turn, a hallmark of neoplasia.
pRB activity is controlled by changes in phosphorylation. pRB is hypophosphorylated in normal quiescent cells (in G
0
phase) and in cells that are in early G
1
. With continued progression through G
1
, cyclin-dependent kinases. (Cdk; Cdk), in association with their respective cyclins, phosphorylate pRB at a number of serine and threonine residues. Unphosphorylated, pRB binds to and sequesters transcription factors of the E2F family. Phosphorylated, pRB discharges these factors, the factors in turn activating transcription of genes coding for proteins regulating DNA replication and cell proliferation. These events commit the cell to entry into S phase. Later, in late mitosis, type 1 protein phosphatases dephosphorylate pRB, restoring the active, E2F-sequestering form, thus resetting the cycle.
pRB is also essential in the terminal differentiation of cells of various lineages. During terminal differentiation, when cells exit the cycle, pRB expression is upregulated and the protein remains in the active—that is, hypophosphorylated—state. Mice homozygously deleted for the RB gene show defective differentiation of various tissues.
Given the critical role that pRB phosphorylation plays in controlling progression of cells through the cell cycle and in mediating terminal differentiation, there exists a need for assays that permit the ready determination of pRB phosphorylation status.
Typically, the phosphorylation status of pRB is assayed in vitro, measuring
32
P-labeling and/or electrophoretic mobility of the protein after isolation and identification by Western blotting. The procedure is cumbersome, and more importantly risks artifactual activation of phosphatases that may dephosphorylate the protein during or after cell lysis. There thus exists a need for methods of measuring pRB phosphorylation in intact cells.
Furthermore, the existing methods measure phosphorylation of pRB in bulk culture. Several questions regarding the mechanism by which pRB controls cell cycle progression cannot be answered using such assays. For example, is phosphorylation of pRB within the cell an all-or-none phenomenon, or is there instead a mixture of hypophosphorylated and hyperphosphorylated pRB molecules at varying proportions throughout the cycle? What proportion of pRB molecules is phosphorylated within the cell during G
1
, prior to entrance to S phase? Is there a critical threshold in the ratio of hypophosphorylated to hyperphosphorylated pRB molecules that determines the transition of cells to quiescence or to commitment to enter S? Is it the ratio of hypo- to hyperphosphorylated pRB or, rather, the total level of the latter that is critical for cell commitment to enter S phase?
Study of the average behavior of cells in bulk culture also precludes evaluation of heterogeneity in the cycling of individual cells in the population. Such heterogeneity in cell cycle kinetics in tumor cell populations is recognized as a major impediment to successful therapy of cancer. There thus exists a need for methods that permit the heterogeneity of cell cycle kinetics to be assayed, and a particular need for methods that would permit cell cycle heterogeneity to be assessed in populations of tumor cells.
The heterogeneity in the cycling kinetics and timing of cells in culture typically obligates artificial synchronization of the cells in culture to permit meaningful results to be obtained using the existing bulk assays; and yet this cell cycle synchronization, when induced by inhibitors of DNA polymerase, is associated with growth imbalance and unscheduled expression of cyclins. Gong et al.,
Cell Growth Differ
. 6:1485-1493 (1995). There thus exists a need for methods that permit the phosphorylation status of pRB to be measured in individual cells without exogenous intervention in the cell cycle.
Methods permitting the phosphorylation status of pRB to be measured in individual cells would prove useful additionally in identifying and characterizing antiproliferative agents that act by halting progression through the cell cycle.
ONCONASE®, initially named protein P30, is a basic protein of 12,000 MW isolated from oocytes or early embryos of
Rana pipiens
. ONCONASE® shows antiproliferative activity in vitro, suppressing proliferation of tumor cell lines of various lineages, including those of hematological origin. ONCONASE® has also been shown to inhibit growth of certain tumors in vivo in mice.
Although ONCONASE® is currently in clinical trials for treatment of patients with advanced pancreatic adenocarcinoma and malignant mesothelioma, the mechanism of its antitumor activity is still poorly understood. The protein is known to have both cytostatic and cytotoxic effects, the former manifesting as an increase in the proportion of cells in G
1
phase of the cell cycle; but the mechanism by which the drug effects such cell cycle arrest is unknown. It would be advantageous to have an assay that would permit such a drug's effects on the cell cycle, and in particular, its effect, if any, on pRB phosphorylation, readily to be assayed on a single-cell basis.
Reliable measures of pRB phosphorylation status in individual cells would permit pRB to serve as a marker for distinguishing quiescent from cycling cells. Although certain cell features, such as cellular RNA content, nucleolar mass, chromatin structure (degree of chromatin condensation), expression of the Ki-67 antigen, or expression other proliferation-associated proteins have been proposed as markers distinguishing cycling from noncycling cells, there is as yet no generally accepted, easily measurable marker which discriminates G
0
from G
1
cells. There thus exists a need in the art for a marker that reliably discriminates quiescent cells from cycling cells.
Recently, two mAbs recognizing human pRB have been described, one of which specifically detects the underphosphorylated form of this protein (pRB
P−
), the other of which reacts with total pRB, regardless of phosphorylation state (pRB
T
). Dunaief et al.,
Cell
79:119-130 (1994); Wang et al.,
Oncogene
8:279-288 (1993); Terada et al.,
J. Immunol
. 147:698-704 (1991); Zarkowska et al.,
Oncogene
14:249-266 (1997). The
Darzynkiewicz Zbigniew
Gruenwald Stefan
Juan Gloria
Traganos Frank
Becton Dickinson and Company
Petry Douglas A.
Saunders David
LandOfFree
Flow cytometric methods for the concurrent detection of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Flow cytometric methods for the concurrent detection of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Flow cytometric methods for the concurrent detection of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3327717