Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-11-30
2001-09-11
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007200, C436S064000
Reexamination Certificate
active
06287790
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the use of nuclear structural proteins, particularly NuMA (Nuclear Mitotic Apparatus Protein) as: a diagnostic indicator of cell phenotype in histopathology; a transporter protein, to which therapeutic agents of interest may be linked for delivery to the nucleus; and a drug discovery target for agents that block or mimic the interaction between NuMA and its binding partners.
BACKGROUND OF THE INVENTION
Abbreviations: CMS protein, connective membrane skeleton protein; FA, focal adhesion; NLS, nuclear localization signal; NES, nuclear export signal. NM, nuclear matrix; rBM, reconstituted basement membrane; HMEC, human mammary epithelial cells; 2D and 3D, two and three dimensional; Rb, retinoblastoma protein; ECM, extracellular matrix; EGF, epidermal growth factor; mAb, monoclonal antibody;
The cell nucleus is organized by a non-chromatin internal structure referred to as the nuclear matrix (NM). Identified NM components include coiled-coil proteins, cell cycle regulators, tissue-specific transcription factors, and RNA splicing factors. Although splicing factors have been shown to redistribute during cellular differentiation, and following the induction of gene expression, such alterations in nuclear organization, defined here as the spatial distribution of nuclear components, are thought to be the consequence of changes in gene expression. However, both NM composition and structure may affect gene expression and cellular function, and thus a systematic analysis of nuclear organization during such a complex process as tissue differentiation is warranted.
To study the effect of cell growth and tissue differentiation on nuclear organization, we have used a reconstituted basement membrane (rBM)-directed model of mammary gland morphogenesis. The HMT-3522 human mammary epithelial cells (HMECs) were isolated from reduction mammoplasty and became immortalized in culture. When embedded within a rBM, these cells growth arrest, organize an endogenous BM and form polarized acinus-like structures with vectorial secretion of sialomucin into a central lumen. Using this model, we have compared the nuclear organization of HMECs cultured on a plastic surface (2D monolayer) vs. a 3-dimensional (3D) rBM. Nuclear organization was assessed by examining the distribution of the coiled-coil NM proteins lamin B and NuMA, the cell cycle regulator Rb (p110Rb; 5), and the splicing factor SRm 160 (formerly known as B 1 C8; 16). These proteins had distinct spatial distribution patterns specific for proliferation, growth-arrest and acini formation. Moreover, disruption of nuclear organization in acini by either perturbing histone acetylation, or directly modifying the distribution of NuMA proteins, altered the acinar phenotype.
We have previously determined that the extracellular matrix (ECM) directs morphogenesis and gene expression in mammary epithelial cells. Here we show that a reciprocal relationship exists between the ECM and nuclear organization. These findings underscore a role for nuclear organization in regulation of gene expression and provide a possible framework for how cell-ECM interactions determine cell and tissue phenotype.
Structure Of NuMA Proteins Used Herein
Nuclear Mitotic Apparatus protein (NuMA) is a 238-kDa protein of the nuclear matrix in interphase that relocates to the spindle poles in mitosis. The globular tail domain (residues 1701 to 2115) contains the nuclear targeting sequence, the site for binding to the mitotic spindle as well as a site responsible for nuclear reformation. In the full-length NuMA molecule, point mutations at position 1988 or 1989 or a double mutation at residues 2004 and 2005 cause NuMA to accumulate in the cytoplasm of both BHK and HeLa cells. It is believed that a bipartite nuclear location signal involving the sequences RKR (1987-1989) and KK (2004-2005) which are separated by 14 amino acid residues is necessary for translocation of NuMA from the cytoplasm. Observation of micronuclei formation suggests that a region important for normal nuclear reformation lies in the C-terminal 130 residues. Finally, NuMA mutant proteins ending at or after residue 1800 bind to the spindle poles of mitotic cells, while NuMA proteins ending at or before residue 1750 do not. The NuMA protein further contains phosphorylation sites. Mutation of the predicted p34cdc2 phosphorylation sites in NuMA impairs the assembly of the mitotic spindle and blocks mitosis. A preferred sequence for NuMA is taken from EMBL GenBank DDBJ: Assession Number Z11584, and is shown in SEQ.ID. No. 1.
SUMMARY OF THE INVNTION
Using a 3D model of human mammary epithelial cell culture, we have shown that the distribution of the structural protein NuMA within the nucleus depends on the cell status, and that the distribution of NuMA into enlarged peripheral foci observed in acini-like structures (characteristic of normal breast tissue) plays a role in maintaining acini phenotype and the state of chromatin organization.
We can disrupt NuMA distribution by introducing antibodies directed against part of the sequence of the protein in living cells (after acini formation) and subsequently alter cell and tissue phenotype. Such phenomenon was not reproduced in breast tumor cells in which NuMA is diffusely distributed in the nucleus.
We also have data showing that NuMA shuttles between nuclear sites and cytoplasmic sites. The fact that NuMA is found associated with the cell skeleton in both the cytoplasm (cytoskeleton) and the nucleus (nuclear matrix- the non chromatin structure of the nucleus) suggests this protein is an anchor protein which may participate in signal transduction by shuttling between specific sites in the cell and initiating cellular functions by itself or via the tethering of other proteins.
These data enable us to conceive and reduce to practice the following methods for utilizing the supramolecular organization of nuclear proteins for detection and treatment of pathologies.
1) Relocalization of nuclear proteins inside the nucleus may be used as a read-out to identify cell phenotype in histopathology.
It has been shown that localization of proteins within a tissue depends on the stage of development and differentiation (shown for extracellular matrix proteins, cell adhesion complex-associated proteins, retinoic acid receptor, topoisomerase II). Moreover variation in protein localization inside a tissue is often associated with changes in expression of the protein. Our work has demonstrated that the distribution of proteins also varies within the nucleus of cells that are part of a morphogenesis and differentiation process and that, in this case, the change in distribution is not associated with an alteration in the level of protein expression. A few examples of redistribution of nuclear proteins were already described in the literature, but none had demonstrated a clear relationship between the progressive relocalization of nuclear proteins and a morphogenic and differentiation process including proliferation, growth-arrest and complete morphogenesis of acini. We have also shown that there is a characteristic organization of NuMA in breast tumor cells and that NuMA organization in revertant cells mimics the organization observed in phenotypically normal cells (-cells arranged into acini).
According to the present invention, the localization of nuclear structural proteins such as NuMA is used to identify tumors cells (starting with mammary tissue) and different stages in the tumor progression and differentiation processes. The technique encompasses immunostaining of cell culture and tissue sections obtained from reduction tissueplasty.
We have shown that the nuclear mitotic apparatus protein (NuMA) redistributes within the nucleus of non-malignant mammary epithelial cells undergoing acinar morphogenesis in a three-dimensional (3D) system of culture. Notably, proliferation, mitotic phase, growth-arrest, and formation of breast glandular structures (acini) are characterized by a different nuclear localization of NuMA. Following differentiatio
Bissell Mina
Lelièvre Sophie
Aston David J.
Caputa Anthony C.
Hunt Jennifer
Nold Charles R.
The Regents of the University of California
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