Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-01-21
2002-10-01
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S467000, C435S366000
Reexamination Certificate
active
06458593
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods of immortalizing primary cells and to the immortalized cell lines produced by these methods.
BACKGROUND OF THE INVENTION
The desirability of generating immortalized cell lines for use as in vitro models of various tissues has long been recognized. For example, researchers have endeavored for some time to generate a suitable in vitro model for cells of the anterior pituitary gland. The anterior pituitary gland of mammals is composed of specialized cells known to synthesize and secrete a variety of hormones that regulate critical body functions. Corticotropes express adrenocorticotropic hormone (ACTH) which regulates the steroid hormone output from the adrenal gland. Gonadotropes express the gonadotropin hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which operate in concert to control reproductive functions. Thyrotropes are the source of thyroid-stimulating hormone (TSH), which regulates the synthesis and release of thyroid hormones from the thyroid gland. Somatotropes express growth hormone (GH), which controls cell growth. Lactotropes express a hormone closely related to growth hormone, prolactin which controls lactation and other functions as well. Prolactin was discovered in 1970 and its entire functional role is not yet understood (Hodson, Calif., 1996). Melanotropes express melanocyte-stimulating hormone (MSH), which regulates pigmentation of the skin. The suffix “troph” may be substituted for “trope” when referring to these cells. Control of end-organs regulated by anterior pituitary hormones is closely regulated by circulating end-organ hormones and neural inputs which interact at pituitary and hypothalamic sites to maintain homeostasis.
Some pituitary cell lines presently exist but these are usually neoplastic cells which continue to divide in vitro by virtue of malignant transformation, e.g., the GH-3 rat cell line was derived from a pituitary tumor. GH-3 cells are termed somatomammotrophs because they synthesize and secrete both prolactin and growth hormone. Other rat pituitary hormone-secreting cell lines are also known, e.g., GH-1, which was derived from the same tumor as the GH-3 line. The RC-4B/C cell line is an apparent mix of corticotropes, thyrotropes, lactotropes and somatotropes. Likewise, with regard to human pituitary cell lines, most of these come from neoplasms of the pituitary. Prolactinoma is the most prevalent form of pituitary neoplasm in man. Therefore, human lactotrope adenoma cells have been extensively studied to determine the genetic mechanisms of tumor formation; such studies suggest that expression of the hst gene encoding fibroblast growth factor may be associated with pituitary tumorigenesis (Cai, W Y, et.al., 1994; Gonsky, R et.al., 1991). Hydridomas formed by fusion of malignant cells with gonadotropes have been used to express human FSH (U.S. Pat. No. 4,383,034). LH has also been produced by fusion of human pituitary adenoma cells with a human lymphoblastoid cell line (U.S. Pat. No. 4,383,035).
Transformed cells are not the same as non-transformed cells present in the body. Transformation shifts the expression of endogenous proteins significantly, turning off the expression of some proteins, while the expression of other proteins is increased. Transformation also alters morphological and cellular properties, e.g. transformed cells require lower amounts of serum and growth factors to support proliferation. Transformed cells may also erroneously process proteins resulting in molecular alteration of endogenous proteins. Human cytomegalovirus immediate-early promoter was more efficient in directing reporter gene expression when expressed in transformed than non-transformed rat anterior pituitary cells suggesting that transformation alters transcriptional factors (Coleman, T A, et.al., 1991). Thus, a transformed pituitary cell may not reflect the normal physiological and cellular processes of its progeny.
Hormone-secreting cells of the pituitary can be grown in vitro as a primary culture. Such primary cultures of pituitary cells have been used extensively to investigate physiological processes involved in regulation of hormone secretion. For example, these cultures were used to bioassay specific hypothalamic releasing hormones that regulate hormone output of the pituitary. This discovery provided considerable insights into the mechanisms of intercommunications between the nervous and endocrine systems (Guillemin, 1978). However, after several cell divisions primary cultures invariably reach a crisis stage and thereafter cease to divide. This property is referred to as senescence. There are some pituitary cell lines which continue to proliferate in culture, but are apparently non-transformed. Mouse thyrotrope tumors such as TtT 97 result from thyroidectomy, synthesize and release TSH, and respond to T3 and TRH (Furth, J, 1955; Condliff, P G, et.al., 1969; Cacicedo, L, et.al., 1981). The rat somatomammotroph cell line, rPCO and clonal derivatives, was derived from primary culture of rat pituitary cells in media containing T3 and GHRH, which may have resulted in selective proliferation of somatotropes. RPCO cells synthesize and release growth hormone and prolactin; secretion is differentially affected by TRH, T3 and GHRH (Chomczynski, P, et.al., 1988; Kashio, Y, et.al., 1990). Both of these cell lines have been used in basic research into the molecular biology of pituitary hormone processing (Coleman, T A, et.al., 1991; Wood, W M, et.al., 1989). The mechanisms by which these cells are immortalized is unknown. These cells have not yet been definitively demonstrated to be non-transformed (Chomczynski, P, et.al., 1988).
Recombinant DNA technology has also been used to produce protein hormones of the pituitary gland. The non-glycosylated monomers of growth hormone and prolactin were initially expressed in
E. coli
cells using a recombinant plasmid containing the cDNA for growth hormone or prolactin. Recombinant protein accumulates in inclusion bodies primarily as reduced monomers. Following solubilization with urea, reoxidized monomers were recovered through use of reoxidation procedures which resulted in biologically active material similar to native hormones with the addition of a methionine residue at the amino terminus (Paris, N, et.al., 1990). The group of heterodimeric glycoprotein hormones including hCG, LH, FSH and TSH have also been expressed in Chinese Hamster Ovarian (CHO) cells using cloned sequences of both the homologous &agr;-subunit and the hormone-specific &bgr;-subunit (Reddy, V B, et.al., 1985; Simon, J A, et.al., 1988; Keen, J L, et.al., 1989; U.S. Pat. No. 4,923,805; U.S. Pat. No. 5,156,957; U.S. Pat. No. 4,840,896). These heterodimeric proteins are known to be glycoproteins containing 15%-35% carbohydrate as N-linked and O-linked glycans present on both the &agr; and &bgr;-subunits. These carbohydrates add structural complexity and have various functions in the assembly, stabilization, modulation of biological activity and control of clearance of these molecules (Szkudkinski, M W, et.al., 1996; Galway, A B, et.al., 1990). The addition of carbohydrate moieties to the protein backbone is a function of the host cell and since these are different from the natural hormone-producing cells, variation in the glycosylation of recombinant hormones may result. Detailed comparison of urinary and recombinant human FSH (rhFSH) has shown that rhFSH is more acidic, suggesting differences in sialic acid content or terminal monosaccharides (de Leeuw, R, et.al., 1996). Also, recombinant human TSH (rhTSH) contains only sialic acid at terminal biantennary monosaccharides while pituitary-derived hTSH also contains sulfated N-acetyl galactosamine terminal residues. This results from the lack of N-acetyl galactosamine transferase in CHO cells used for the expression of rhTSH (Szkudlinski, M W, et.al., 1996).
There are non-transformed cells in the art which are thought to arise through spontaneous immortalization as occurs in non-malignant or benign tumors. How
Burnett Bruce P.
Gill, IV John Charles
Hedlund Tammy E.
Musick James R.
Ketter James
Sheridan & Ross P.C.
Vitro Diagnostics, Inc.
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