CYCLIC NUCLEOTIDES, CALCIUM, BASE ANALOGS, AND TRANSFORMATION IN VITRO

29 May

In Chapter 14, modulation of cell transformation in vitro was discussed. The effects of a variety of structurally dissimilar chemicals in modulating the transformation process were listed in Ta- ble 14.9. However, several such chemical modulators of the transformation  phenotype in vitro were purposely omitted to be discussed in this chapter, primarily because their effects can be closely related to other mechanisms, and in some instances a fair amount of knowledge exists as to the mechanism of the effects themselves. Each of these three factors is considered separately.

Cyclic Nucleotides and Cell Transformation

A possible role for cyclic nucleotides in cell transformation  in vitro was suggested some three decades ago when studies by Pastan and associates (cf. Johnson et al., 1971) demonstrated that the addition of cyclic AMP to CEF cultures transformed by the Rous sarcoma virus (RSV) re- sulted in an alteration in their morphology to that of normal, contact-inhibited cells with a lower growth  rate. However,  further investigation  demonstrated  that not all transformed  cells re- sponded in this manner upon the addition of cyclic AMP to the medium (Roth et al., 1982). The concentration of cyclic AMP in cells transformed in vitro was significantly less than that in cor- responding nontransformed cells, especially during conditions of little or no cell replication (cf. Pastan, 1975). However, at least in cell lines transformed  by temperature-sensitive  SV40 mu- tants, a change in temperature from the restrictive to the permissive temperature or vice versa did not alter the cyclic AMP levels of these cells (Burstin et al., 1974). Otten et al. (1971) demon- strated an inverse correlation between growth rate and cyclic AMP levels when many cell lines were compared during logarithmic growth. Furthermore, in RSV-transformed  chick embryo fi- broblasts, plasma membrane  adenylate cyclase activity was reduced, and its Km for ATP was

Figure 16.4 Computer  interpretation  of the [35S]-methionine-labeled proteins (IEF) secreted by normal human fibro- blasts (MRC-5). Polypeptides present preferentially in the media from SV40-transformed  fibroblasts have been included in the map and are indicated in black. (Adapted from Celis et al., 1987, with permission of the authors and publisher.)

significantly  lower in normal cells than in the transformed  cells. Anderson  et al. (1973) sug- gested that these changes may be mediated through some modification of the plasma membrane by viral transformation. Other studies by Sharma and associates (1977) as well as by Simantov and Sachs (1975) demonstrated apparent structural changes in cyclic AMP–binding proteins in an adrenocortical carcinoma and neuroblastoma, respectively.

Alterations in the cyclic AMP levels in normal and transformed cells have also been corre- lated with changes associated with the plasma membrane. Willingham and Pastan (1974) dem- onstrated  that, in cultured  mouse 3T3 cells, low levels of intracellular  cyclic AMP may be correlated with increased agglutinability  by concanavalin  A, whereas high levels of the cyclic nucleotide are seen in those cells exhibiting decreased agglutinability. Cyclic AMP also appears to affect the glycopeptide composition of the surface membranes of cultured cells (Roberts et al.,

1973), and the morphological changes seen in cultured neoplastic cells after the addition of cy- clic nucleotides are also associated with a reappearance of contact inhibition of growth in these cells (cf. Pastan, 1975).

Another cyclic nucleotide, cyclic GMP, was shown by Goldberg and associates (see Had- den et al., 1972) to vary with cell replication  in a manner opposite  to that seen with cyclic AMP—that  is, cyclic  GMP levels  increase  when lymphocytes  are stimulated  to replicate, whereas the levels of this cyclic nucleotide are decreased in starved cells, in which cyclic AMP levels increase. In embryonic hamster kidney fibroblasts in culture, the addition of serum, insu- lin, or other growth factors caused a decrease in cyclic AMP levels but had no effect on cyclic GMP levels. However,  when these cells were transformed  by polyomavirus,  the cyclic GMP concentration  increased  sixfold (Richman  et al., 1981). The inverse relation between  cyclic AMP levels in cells and their rate of replication has led to applications both in experimental and clinical situations. Bang et al. (1994) has shown the induction of terminal differentiation by ex- ternally added cAMP to a line of cells derived from adenocarcinoma  of the prostate in the hu- man. The addition  induced  terminal  differentiation  of these cells in vitro. In addition,  Cho- Chung and associates (cf. Tagliaferri et al., 1988), using analogs of cAMP, induced both growth inhibition and phenotypic reversion of murine sarcoma virus–transformed  mouse 3T3 cells. A more extensive review by these investigators  laid the foundation  for potential clinical uses of such chemicals in the therapy of neoplasia (Cho-Chung et al., 1991).

Calcium Ions and the Neoplastic Transformation in Vitro

Already discussed is the importance of calcium ions in activating protein kinase C, an enzyme that appears to be important in the mediation of some tumor promoters  (Chapters 7 and 15). Calcium ions were shown to be very important in mediating the action of cyclic AMP in a vari- ety of functions (cf. Rasmussen,  1974; Whitfield et al., 1979). It is now apparent that a large number of intracellular Ca2+-binding proteins are involved in signaling mechanisms (cf. Niki et al., 1996). One of the most important of these proteins is calmodulin, which is involved in the regulation of protein phosphorylation–dependent cascades as well as in interaction with cyto- skeletal  elements  (cf. Veigl et al., 1984). Generally  speaking,  the calmodulin  levels of trans-

formed cells of mesenchymal origin are higher than those present in nontransformed  cells (cf. Veigl et al., 1984). Early studies showed that both mouse and chicken fibroblasts transformed by oncogenic viruses, as compared with their normal counterparts, required a markedly decreased

concentration  of Ca2+ in the medium to sustain cell replication in culture (Boynton and Whit-

field, 1976; Balk et al., 1979). This was also found to be true for keratinocytes, in which high concentrations  of calcium induce differentiation  and ultimately  apoptosis,  but low concentra- tions of the ion favor keratinocyte replication. Transformed keratinocytes could not be induced to differentiate in high concentrations of calcium ions and, in fact, replicated quite well in low concentrations (cf. Whitfield, 1992). Although the mechanisms of many of these differences are not absolutely clear, it is now apparent that cells not only have specific Ca2+ channels for en- trance but also Ca2+-sensing receptors. This is diagrammed in Figure 16.5, which also indicates the importance of the endoplasmic reticulum as a transporter and storehouse for calcium within cells (cf. Hebert and Brown, 1995). Thus, it is likely that transformed cells that do not respond to calcium in normal ways have defects in one or more of the pathways shown in Figure 16.5 as well as in the regulation  of the expression  of various  calcium-binding  proteins,  especially

calmodulin.

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