In Chapter 13, the use of the thymine analog 5-bromodeoxyuridine was discussed in relation to the demonstration of sister chromatid exchanges (Figures 13.5 and 13.6). Almost 30 years ago, Silagi (1971) demonstrated an interesting effect of the incorporation of 5-bromodeoxyuridine into the DNA of mouse melanoma cells. Growth of these cells in media containing the analog caused a suppression of growth, loss of pigment formation, and a lost or markedly decreased ability to grow as tumors when inoculated into appropriate hosts. These effects were completely
Figure 16.5 Diagram illustrating the manner in which Ca2+ acts as an extracellular messenger. Activa- tion of the Ca2+-sensing receptor (by binding of Ca2+ to negatively charged regions) activates phospholi- pase C (PLC; possibly via a Gq protein), leading to increased intracellular levels of diacylglycerol (DG) and
inositol 1,4,5-trisphosphate (IP3), and concomitant release of Ca2+ from internal stores (e.g., the endoplas-mic reticulum). The rise in Ca2+ is sustained by influx of Ca2+ through channels in the plasma membrane.
The Ca2+-sensing receptor can also reduce receptor-mediated increases in cAMP levels (possibly via a Gi protein). The Ca2+-induced changes in the activities of these second messenger systems leads to changes in the activities of a series of kinases (e.g., PKC and PKA), which in turn alter the biological activities of the cell. AC, adenylate cyclase; PIP2, phosphatidylinositol bisphosphate. (From Hebert and Brown, 1995, with permission of the authors and publisher.)
reversed by growing the cells in thymine without 5-bromodeoxyuridine. This was of great inter- est, since—in addition to the induction of sister chromatid exchange—5-bromodeoxyuridine also induces specific-locus mutations and the expression of fragile sites in cultured cells (Morris,
1991). More recently, Anisimov and associates (1993) demonstrated the carcinogenicity of this analog in association with other agents when administered in vivo for chronic periods. Other effects of addition of this analog to the medium of cultured cells include the induction of endog- enous oncogenic viruses, especially RNA viruses (cf. Goz, 1978). In a sense, it would appear that the analog tends to enhance “differentiation” of the expression of the viral genome.
In addition to 5-bromodeoxyuridine, 5-azacytidine, an analog of cytidine, also has the ca- pacity to “activate” retroviral genomes within a variety of cells in vitro (Jaenisch et al., 1985). In addition, in other systems the analog induces the appearance of differentiated characteristics in a variety of different cell types (Jones, 1985). It is presumed that the effects of this analog are due to its effects on DNA methyltransferase responsible for the methylation of cytosines in DNA (Jüttermann et al., 1994). However, exactly how the 5-bromodeoxyuridine exerts its effect on reversing neoplasia and altering differentiation is not clear at the present time.
GROWTH FACTORS AND THE NEOPLASTIC TRANSFORMATION
Biologists have long recognized the presence and critical importance of factors, both exogenous and endogenous, capable of regulating either positively or negatively the growth, development, and cell replication of cell populations within an organism. Paramount among these factors are the trophic hormones of endocrine origin, such as those from the anterior pituitary, pancreatic islets, and gonads. However, the importance of trophic factors in regulating cell growth and replication was not entirely appreciated until the advent of tissue culture methodology. The crit- ical importance of serum in the maintenance, growth, and replication of mammalian cells in cul- ture was obvious with the very earliest experiments of Carrel and others (cf. Carrel and Lindbergh, 1938).
Serum and the Nutrition of Transformed Cells
One of the earliest studies showing distinctive differential serum requirements for transformed cells was that of Temin (1966), who demonstrated a lowered requirement for serum by cells transformed by the RSV compared with nontransformed chick embryo fibroblasts. Similarly, SV40-transformed human lung fibroblasts exhibited a lowered requirement for a number of se- rum factors including some ions (McKeehan et al., 1981). Chemically transformed 3T3 mouse cells also had a much lower serum requirement than normal cells, although these cells exhibited “normal” growth controls at low serum concentrations (Holley et al., 1976).
In contrast to the lowered serum requirement of transformed cells, a number of publica- tions have indicated that neoplastic cells in culture exhibit an increased requirement for the es- sential amino acid methionine (Halpern et al., 1974; Hoffman, 1982; Breillout et al., 1990). Studies discussed in Chapter 17 also indicate that certain neoplasms have specific requirements for other individual amino acids. With this knowledge, it has also been possible to develop se- rum-free cell culture media for both normal and neoplastic cells (cf. Taub, 1990; Bjare, 1992). However, the development of such serum-free media initially required the isolation and charac- terization of factors—virtually all polypeptide in nature—occurring in serum and having both trophic and inhibitory effects on cells in culture. A number of these growth factors and related agents are discussed below.