Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1998-04-17
2001-06-12
Hutzell, Paula K. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S388100, C530S387100
Reexamination Certificate
active
06245899
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
This invention is related to therapeutics and the regulation of cellular growth and differentiated function. More specifically, it relates to cell density signal molecules produced by fibroblastic cells (for example, tendon cells), to the antibodies which recognize them, and to the use of these molecules in the treatment and repair of connective tissue injury disease, and regulation of cellular proliferation.
2. Description of Related Art
The level of collagen expression, a differentiated function of some fibroblastic cells in culture, is under the regulation of several environmental factors, one of which is cell density. Beginning in the early 1960's investigators observed that an enhancement of collagen production was dependent upon an increase in cell density; but, despite its long history, little is known about the signaling mechanism that allows the cell to recognize the presence of its neighbors and translate this information into increased collagen synthesis.
Most cell types which naturally express procollagen in vivo lose this ability over time when placed in cell cultures. However, primary avian tendon (PAT) cells, when grown in a cell culture environment that is permissive for high procollagen expression, retain the potential for high levels of collagen expression, and, in this regard, demonstrate a sensitivity to the density of the cell culture. PAT cells increase their production of procollagen, in direct relation to cell density, from less than 10% to about 50% of total protein synthesis. See for example, Schwarz, R. I. and M. J. Bissell,
Proc. Nat. Acad. Sci.
(1977) 74:4453-4457.
The proliferative capacity of cells in culture is also affected by cell density. However, a definitive correlation has been difficult to obtain because cell proliferation is affected by many cell culture parameters, only one of which is cell density.
For instance, it is difficult to distinguish the effect of density per se from the possibility that cell density changes are a subset of the nutritional needs of the cell. Similarly, decreased access to growth factors as cells lay down an extracellular matrix might explain the cell density effect. Changing the medium has been shown to be sufficient to stimulate cell division. Even just shaking the medium above the cells can stimulate cells to divide (Stoker, M. and D. Piggott,
Cell
(1974) 3:207-215).
Cell contact may also play a role as demonstrated by the fact that membrane components, shed into the medium, act to inhibit cell proliferation.
Consequently, the relationship between cell density on the one hand and cell proliferation and the expression of differentiated function (procollagen gene expression in the case of PAT cells) on the other hand, has been a complex problem for which it has been particularly difficult to design definitive experiments.
Despite these complications, several publications describe proteins which are reported to affect density dependant cell growth. For example a 40-45 kD density-dependent growth inhibitor (designated IDF-45), isolated from mouse 3T3 cells has been described in Harel, L., et al.,
J. Cell. Physiol.
(1984) 119:101-106; Harel, L., et al.,
J. Cell. Physiol.
(1985) 123:139-143; and Blat, C., et al.,
J. Cell. Physiol.
(1987) 130:416-419.
Lipkin, G., et al. (
Cancer Res.
(1978) 38:635-643), and Fass, E., et al. (
J. Invest. Dermitol.
(1986) 87:309-312) disclose a contact inhibitory factor, isolated from hamster melanocytic cells, which restores density-dependent growth to melanoma cells.
Yaoi, Y. and K. Motohashi (
GANN Monograph on Cancer Res.
(1980) 25:29-39) disclose a low molecular weight (6-8 kD) growth inhibitory factor from the cell surface of chick embryo fibroblasts.
The subject invention discloses a proteinaceous cell density signal molecule, exhibiting a molecular weight of at least about 25 kD to at most about 35 kD, most preferably about 30 kD, which: 1) first associates with the extracellular matrix of PAT cells in culture; 2) transiently stimulates the proliferation of these cells; and, 3) subsequently stimulates procollagen gene expression.
SUMMARY OF THE INVENTION
A proteinaceous cell density signal (CDS) molecule has now been found. The CDS is capable of temporarily stimulating the proliferation of fibroblastic cells in culture and thereafter promoting differentiated gene expression. The CDS is secreted by fibroblastic cells, for example, tendon cells, and can be obtained free of such cells. If desired, it can be substantially purified.
This novel polypeptide is secreted by fibroblastic cells, such as tendon cells, and provides a means by which the cells self-regulate their proliferation and the expression of differentiated function. The CDS, and the antibodies which recognize it, are important for the development of diagnostics and treatments for injuries and diseases involving connective tissues, particularly tendon.
More particularly, naturally occurring avian CDS is a 25 kD to 35 kD growth stimulatory polypeptide, expressed by primary avian (chicken) tendon cells in culture, which is believed to quickly bind to the extracellular matrix (ECM) produced by these cells. This quick binding is believed to restrict the diffusion of CDS and thereby proximally limit the proliferative effect of this molecule.
At some time after the binding of CDS to the ECM, a subsequent event—which is not presently understood—occurs such that the activity of CDS is altered and it becomes stimulatory for the expression of procollagen, a differentiated function of the avian tendon cells.
CDS may be harvested from cultures of tendon cells by gentle agitation. This treatment removes a substantial fraction of CDS from the cell layer and cells which have been so treated exhibit reduced expression of procollagen. CDS, so harvested, exhibits growth stimulatory activity when added back to cultures of quiescent tendon cells. This surprising and unique characteristic of CDS—that both its removal from and addition to cultures of tendon cells is growth stimulatory—is a feature which distinguishes this polypeptide.
The harvested CDS described above is found in the medium as a multi-molecular complex with an apparent molecular size of at least greater than 30 kD, more usually greater than 60 kD, and most usually approximately 100 kD. After treatment with a sulfhydryl bond disrupting agent, such as DTT, a monomeric, approximately 30 kD, CDS molecule can be identified.
One aspect of the invention is directed to isolated, purified, naturally occurring CDS, and fragments, mutations and modifications thereof which retain CDS biological characteristics.
Another aspect of the invention are antibodies which are specific for CDS.
Still another aspect of the invention are methods of obtaining, isolating and purifying CDS.
Yet another aspect of the invention are methods of using CDS for the therapeutic treatment of injuries to, and diseases of, connective tissue, especially tendon.
REFERENCES:
Martis et al. In Vitro Cellular & Developmental Biology. 22, 241-246, May 1996.
Hutzell Paula K.
Sun-Hoffman Lin
The Regents of the University of California
Verny Hana
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