Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
2000-10-12
2002-04-09
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S004000, C435S029000
Reexamination Certificate
active
06368818
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to cell biology and oncology. More specifically, this invention relates to the methods for visualizing cellular organelles (such as a centrosome) and/or cytoskeletons (such as microtubules) through the use of crystallizing agents (such as tetrazolium salts), to a kit containing crystallizing agents adapted for such use, and to methods particularly for detecting neoplastic cells in a tissue sample, suspension, or a fluid sample by examining the cells for abnormalities in the size, number and shape of cellular organelles (such as a centrosome) and/or cytoskeletons (such as microtubules).
BACKGROUND OF THE INVENTION
The characteristics and functions of cells are determined and maintained by cellular organelles and the cellular cytoskeleton. Cellular organelles include, but are not limited to, nucleus, mitochondria, peroxisomes, Golgi apparatus, lysosomes, endoplasmic reticulum, centrosome, and vacuoles. The term cytoskeleton (cytoskeletal structures) refers to an extensive scaffolding of fibrillar elements, including the three filamentous systems: microfilaments (or actin filaments), microtubules, and intermediate filaments. It may also include linin filaments. The components of the cytoskeleton are involved in diverse cellular functions ranging from mitosis to cell motility to signal transduction. Among these organelles and cytoskeletal structures, centrosome, microtubules, mitochondrion, endoplasmic reticulum, lysosomes, and nuclear envelope are most important.
The centrosome, a central body (or the major microtubule-organizing center (MTOC) of the cell) plays a key role in the temporal and spatial distribution of the interphasic and mitotic microtubule network. Therefore, the centrosome could be considered a major determinant of the overall organization of the cytoplasm and of the fidelity of cell division (Hsu, L. C. and White, R. L. (1998)
Proc Natl Acad Sci U S A
27;95(22):12983-8). Cytoplasmic organization, cell polarity and the equal partition of chromosomes into daughter cells at the time of cell division, once and only once in each cell cycle, are all ensured through the actions of tightly regulated centrosomal function (Tanaka, T., et al., (1999)
Cancer Res
59(9): 2041-4). Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis, and determines the number, polarity, and organization of interphase and mitotic microtubules (Tanaka, T., et al., (1999)
Cancer Res
59(9): 2041-4; Pihan, G. A., et al., (1998)
Cancer Res
58(17):3974-85). The main function of the centrosome is the nucleation of microtubules, and the controlled cycle of its duplication, the two duplicated entities functioning as mitotic spindle poles during subsequent cell division. Centrosomes and their associated microtubules direct events during mitosis and control the organization of animal cell structures and movement during interphase. Although the precise mechanisms by which duplicated chromosomes are equally segregated during mitosis are largely unknown, the centrosome is believed to play an important role(s) in the formation of bipolar spindles (Tanaka, T., et al., (1999)
Cancer Res
58(17):3974-85). The microtubule nucleating ability of centrosomes of tissue sections is retained even after several years of storage as frozen tissue blocks (Salisbury, J. L., et al., (1999) J. Histochem. Cytochem. 47(10):1265-74).
In animal cells, the centrosome is composed of two centrioles surrounded by the so-called pericentriolar material (PCM), which consists of a complex thin filament network and two sets of appendages.
Malignant tumors generally display abnormal centrosome profiles, characterized by an increase in size and number of centrosomes, by their irregular distribution, abnormal structure, aberrant protein phosphorylation, and by increased microtubule nucleating capacity in comparison to centrosomes of normal tissues (Lingle, W. L. et al., (1998)
Proc Natl Acad Sci U S A
95(6): 2950-5; Xu, X., et al., (1999)
Mol Cell
3(3):389-95; Brinkley, B. R., et al., (1998)
Cell Motil Cytoskeleton
41(4):281-8; Doxsey, S. (1998)
Nat Genet
20(2):104-6; Kuo, K. K., et al., (2000)
Hepatology
31(1):59-64). Among the abnormalities, centrosome hyperamplification is found to be more frequent in a variety of tumor types (Carroll, P. E., et al., (1999)
Oncogene
18;18(11):1935-44; Hinchcliffe, E. H., et al., (1999)
Science
283(5403):851-4; Xu, X., et al., (1999)
Mol Cell
3(3):389-95).
Centrosome consists of many key proteins such as, SKP1p, cyclin-dependent kinase 2-cyclin E (Cdk2-E) (Hinchcliffe, E. H., et al., (1999)
Science
283(5403): 851-4), kendrin (Flory, M. R., et al., (2000)
Proc Natl Acad Sci U S A
23;97(11):5919-23), Protein kinase C-theta (Passalacqua, M., et al., (1999)
Biochem J
337(Pt 1): 113-8), EB1 protein. Recently, a variety of cell cycle-regulated kinases or tumor suppressor genes are located in or are core components of the centrosome. They include Nek2 (Fry, A. M., et al., (1999)
J Biol Chem
274(23): 1304-10), protein kinase A type II isozymes (Keryer, G., et al., (1999)
Exp Cell Res
249(1):131-146), heat shock Cognate 70 (HSC70) (Bakkenist, C. J., et al., (1999)
Cancer Res
59(17):4219-21), PH33 (Nakadai, T., et al., (1999)
J Cell Sci
112 (Pt9):1353-64), AIKs (Kimura, M., et al., (1999)
J Biol Chem
274(11)7334-40), human SCF(SKP2) subunit p19(SKP1) (Gstaiger, M., et al., (1999)
Exp Cell Res
247(2)554-62), STK15/BTAK (Zhou, H., et al., (1998)
Nat Genet
20(2): 189-93), C-Nap1 (Fry, A. M., et al., (1998)
J Cell Biol
274(23): 1304-10), Tau-like proteins (Cross, D., et al., (1996)
Exp Cell Res
229(2):378-87), cyclin E (Carroll, P. E., et al., (1999; Mussman, J. G., et al., (2000)
Oncogene
23;19(13):1635-46), p53, retinoblastoma protein pRB and BRCA1(Hsu, L. C., et al., (1998)
Proc Natl Acad Sci U S A
95(22):12983-8). These proteins are required in the initiation of DNA replication and mitotic progression (Gstaiger, M., et al., (1999)
Exp Cell Res
15;247(2):554-62).
Microtubules, a filamentous system, are linear polymers of alpha- and beta (the beta1, beta2, and beta4 isotypes)-tubulin heterodimers. Except for being a frame of cellular membrane and organelles, microtubules may play an important role in other aspects. Microtubules are involved in diverse cellular functions ranging from mitosis to cell motility to signal transduction. Microtubules are the major constituents of mitotic spindles, which are essential for the separation of chromosomes during mitosis (Shan, B., et al., (1999)
Proc Natl Acad Sci U S A
96(10):5686-5691). They are nucleated by centrosome through the kinetochores of the centrosome. The spindle is a microtubule-based superstructure that assembles during mitosis to separate replicated DNA. Chromosome attachment to and movement on the spindle is intimately tied to the dynamics of microtubule polymerization and depolymerization. The sister chromatid pairs must maintain a stable attachment to spindle microtubules as the microtubules interconvert between growing and shrinking states. Drugs that are currently used in cancer therapy were designed to perturb microtubule shortening (depolymerization) or lengthening (polymerization) (Compton, D. A., et al., (1999)
Science
286:913-914).
Other cytoskeletons such as membrane skeleton, microvilli, cilia, flagella, microfilaments, actin filaments, contractile ring, and intermediate filaments are all important in the organization of the cytoplasm and of the fidelity of cell division.
In addition to the centrosome and microtubules, other cellular organelles or cellular sub organelles such as mitochondrion, chromosomes, chromatin, nuclei, nuclear matrix, nuclear lamina, core filaments, nuclear envelope (NEs), nuclear pore complexes (NPCs), nuclear membrane, centrioles, pericentriolar material (PCM), mitotic spindle, spindle pole bodies (SPBs), contractile rings, proteasomes, telomere, plasma membranes, Golgi complexes, Golgi apparatus, endoplasmic reticulum (ER), endosomes, peroxisomes, proteasomes, phagosomes, riboso
Ozga Brett
Sheridan & Ross P.C.
Witz Jean C.
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