DNA METHYLATION, TELOMERASE, CELL SENESCENCE, AND TRANSFORMATION

29 May

In Chapter 15 the importance of alterations in DNA methylation was discussed, as well as telo- merase activity and telomere length as common alterations in neoplasia. In cultured cells, these functions appear to play a quite significant  role, especially  in the phenomenon  of cell senes- cence, which was briefly described in Chapter 14. In short, essentially all cells that are placed in culture have a finite period or finite number of doublings during which they remain structurally and genetically  normal cells (Hayflick,  1997). At the end of this time, the cells either die or some of them become “transformed”  to an immortal phenotype that is accompanied  by an al- tered karyotype, which usually expresses instability subsequently  as the cells are continued in culture. In recent years, the association of DNA methylation and telomerase and telomeres has been a subject of intense investigation.  These are considered separately here, although in fact they are closely related functions.

DNA Methylation and Transformation

When cells are first placed into culture, there is usually a rapid general loss of DNA methylation; but as immortal cells emerge, the methylated DNA content of such cell lines appears to increase (cf. Razin and Cedar, 1991). Recall that in neoplastic cells in vivo there is a general hypomethy- lation of DNA, although there are clearly areas of hypermethylation within the DNA of a variety of human neoplasms (cf. Laird and Jaenisch, 1994). Baylin and associates (1997) have reviewed some of the changes that may be seen in the DNA methylation capability of cells in culture as a function of the level of the DNA-methyltransferase  activity. Figure 16.15 summarizes much of the experimental data for the relations between DNA methyltransferase activity to fibroblast ag- ing and senescence as well as infection with the SV40 oncogenic virus. The changes in methyla- tion seen on initial explantation are not appreciated from the graph, although the relative DNA methyltransferase  of young fibroblasts may be considered as reflecting the initial loss of DNA methyl groups and, presumably, the activity of the enzyme, especially in epithelial cell cultures. The further loss of methyltransferase as the fibroblasts are continued in culture is reflective of a further loss in DNA methylation  as well. At the time of senescence there is a reversal of this effect, occurring over a shorter time period; at the time of crisis—i.e., in this case transformation by SV40 virus—DNA methyltransferase is subsequently enhanced and then stabilizes. This was further investigated by Slack et al. (1999), who showed that T-antigen expression in transformed cells is accompanied by an elevation in DNA methyltransferase  mRNA and protein as well as global genomic DNA methylation. These results and others (e.g., Kautiainen and Jones, 1986) suggest that transformed cells in cell culture may actually have enhanced levels of DNA methy- lation, which is an interesting contrast to the global demethylation seen in neoplasms in vivo.

Telomeres, Telomerase, and Cell Senescence and Transformation

As suggested in Chapter 15, the normal adult mammal tends to have extremely short or virtually absent telomeres in many of the highly differentiated cell types within the organism. When cells are initially placed in culture, especially mesenchymal cells, they initially have telomeres as well as exhibit telomerase activity. As mesenchymal cells are maintained in culture, their telomeres decrease in length until they become extremely short; the cells then stop replicating and subse- quently die or go through a crisis (see above), at which time immortal cells develop. These im- mortal cells now exhibit telomerase activity and have telomeres, although usually shorter than those in the original cell population  at explantation.  Transformation  by oncogenic  viruses or

Figure 16.15 Graphical  summary of experimental  data for the relations of DNA-methyltransferase (MTase) activity to fibro- blast aging and infection with SV40 virus in vitro. The graph depicts the full changes in DNA-MTase activity relative to 1.0 as the level seen in normal cells between passages 0 and 25. The three components  of the graph are (1) as a function of young human fibroblasts from about passage 25 to when they are senescing (passage 50); (2) cells in the precrisis stage from about passage 50 to 70 after infection with SV40 at about passage 30; and (3) crisis phase cells from about passage 70 to 90, finally immortalized  at passages 130 to 200. (Adapted from Baylin et al., 1998, with permission of the authors and publisher.)

chemicals  will also reactivate  telomerase  and in many instances allow for the immortality  of such transformed cells, as noted in Chapter 14 (Table 14.1). A diagram of this process is given in Figure 16.16.

It should be noted from the figure that germ cells and stem cells have a constant and rela- tively high level of telomerase activity and possess telomeres of substantial size. Induced differ- entiation of immortal cells causes a pronounced downregulation of telomerase activity in a wide variety of transformed  cells (Sharma et al., 1995) but a lack of effect in some other cell lines (Bestilny et al., 1996). There is also evidence that the p53 gene is involved in the entrance of cells into the senescent period (Wynford-Thomas,  1996). The decrease in telomerase activity as

Figure 16.16 The telomere  hypothesis  of cell aging. TRF is the terminal  restriction  fragment  length made up of the terminal TTAGGG repeats in human cells plus a small, variable length, proximal fragment. Germ cells maintain telomerase activity as well as their telomere length upon cell division. Somatic cells in general are telomerase negative and, as a consequence,  their telomeres shorten with each cell division by approximately  50 bp (cf. Krupp et al., 2000). When somatic cell telomeres shorten sufficiently,  cell cycle arrest occurs, with the activation of a senescent phenotype (M1 or Hayflick limit). If cells are transformed, they may bypass  M1, but their telomeres  continue  to shorten,  since telomerase  has not been activated. When telomeres  shorten to the point where chromosome  function is impaired,  cells enter a crisis period (Figure 16.15 or M2). In the absence of transforming  virus infection, a few cells emerge from crisis that have activated the telomere maintenance machinery and telomerase. It is these cells that are immortal and maintain stable telomere lengths during culture. (Adapted from Morin, 1997, with permission of the author and publisher.)

cells in vitro approach senescence does not appear to be a direct function of DNA methylation, although this process can contribute to the repression of telomerase in some cells (Dessain et al.,

2000). The marked shortening of telomeres as cells approach senescence is associated with in- creasing chromosome instability in mammals (Pommier et al., 1995) as well as in lower forms, where inhibition of telomerase  induces chromosomal  destabilization  (Izbicka et al., 1999). In fact, Reddel (2000) has emphasized  that many of the more common genetic changes seen in neoplasia may play a key role in the immortalization process of cells transformed in vitro.

Thus, while there is reasonable evidence to argue that telomerase activity as well as telo- mere length is associated  with aging and senescence  in vitro and in many instances  in vivo (Krupp et al., 2000), the presence of the enzyme and of telomeres, even shortened, has not been universal in neoplasia (Krupp et al., 2000). Still, inhibition of telomerase may be an appropriate method with which to induce death in appropriate neoplasms (Herbert et al., 1999).

THE MOLECULAR  AND CELL BIOLOGY  OF CELL TRANSFORMATION IN VITRO—ITS  CONTRIBUTION  TO OUR UNDERSTANDING  OF THE NEOPLASTIC  PROCESS

As noted in Chapter 14 as well as in this chapter, cells in culture have many but not all of the characteristics  of neoplastic  cells in vivo. In particular,  the cell and molecular  alterations  in transformed cells sometimes appear to be relatively unique to the situation. However, with care- ful comparison  between  normal and transformed  cells in vitro, some characteristics  seen in transformed  cells may be directly applied to the in vivo situation. This was certainly the case with telomerase  and its disappearance  in senescence  and reappearance  during transformation, thus making it a potential therapeutic target in the management of neoplastic disease. Although the multistage nature of neoplasia has not been extensively studied in cell culture, probably be- cause of the lack of good model systems, the SHE cell system has shown that there are consider- able similarities in the biology and quite likely the cell and molecular changes occurring during the three stages of carcinogenesis in this in vitro system. Cell culture offers an exciting tool with which to study the neoplastic transformation and a variety of other processes that impinge on our knowledge and ultimate control of the neoplastic process.

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