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The stage of progression usually develops from cells in the stage of promotion but may develop directly from normal cells as a result of the administration of relatively high, cytotoxic doses of complete carcinogenic agents capable of inducing both initiation and progression. In addition, the incorporation into the genome of genetic information such as oncogenic viruses, the stable transfection of genetic material, or spontaneous chromosomal alterations may enhance the tran- sition into the stage of progression. As previously noted in Table 9.1 and reemphasized in Table

9.3, the principal hallmark and characteristic of the stage of progression is that of evolving kary- otypic instability. It is this molecular characteristic of cells in the stage of progression that poten- tially leads to multiple “stages” or changes in malignant cells that were first described by Foulds (1954) as “independent characteristics.”  As has been pointed out (cf. Harris, 1991), karyotypic changes are common if not ubiquitous in neoplastic cells in the stage of progression. A number of studies (Tlsty et al., 1993; Mäkelä and Alitalo, 1986; Sager et al., 1985) have emphasized the

Table 9.3 Some Cell and Molecular Mechanisms in the Stage of Progression

Genetic macrolesions (chromosomal translocations, deletions, gene amplification, recombination, gene conversion) resulting from evolving karyotypic instability

Irreversible changes in gene expression including fetal gene expression, altered MHC gene expression, and ectopic hormone production

Selection of neoplastic cells with genotypes/phenotypes for optimal growth in response to the cellular environment

Mutations in both alleles of one or more tumor suppressor genes

enhancement of gene amplification in neoplastic cells. Tlsty et al. (1989) demonstrated that the spontaneous rate of gene amplification in a number of neoplastic cell lines was almost 100 times the rate measured in nonneoplastic cells. In contrast, the frequencies of spontaneous point muta- tions have not generally  been significantly  different between normal and neoplastic  cells (cf. Harris, 1991). Recently,  however,  with the demonstration  that alterations  in mismatch  repair genes were responsible for increased incidences of several different types of neoplasms in af- fected individuals (Chapter 5), studies of micro- and minisatellite mutations have been under- taken in a variety of different  neoplasms  (Lothe, 1997; Speicher,  1995). This has led to the finding that, although 50% to 100% of neoplasms with mutations in mismatch repair genes ex- hibit mutations in micro- and minisatellites (Lothe, 1997), approximately 4% to 20% of sponta- neous neoplasms of a variety of types exhibit similar “instability” (Speicher, 1995). The impact of this latter finding is softened by the demonstration that the frequency of microsatellite alter-ations in normal tissue is of the order of 1–5 × 10–2, while that of the variation in minisatellite sequences may be as high as 10–1 in normal human tissues (Simpson, 1997). Of perhaps greater significance in the relationship of microsatellite mutations to the stage of progression is the pro- posal by Shibata et al. (1996) that the diversity of microsatellite mutations may be related to the number of replications  and clonal expansions  of the neoplasm  during its development  in the stage of progression.

Mutations in proto-oncogenes  and tumor suppressor genes are relatively common in ma- lignant neoplasms, both in the human (Yokota and Sugimura, 1993; Kiaris and Spandidos, 1995; Harris, 1996) and in the animal (Stowers et al., 1987; Jacks, 1996). In general, mutations can be identified in proto-oncogenes at early stages, including preneoplasia in the development of neo- plastic disease (Burmer and Loeb, 1989; Ando et al., 1991; Ranaldi et al., 1995; Pellegata et al.,1994; Bauer-Hofmann  et al., 1992). While many but not all proto-oncogene  mutations can be seen in very early lesions, mutations in the p53 tumor suppressor gene in a variety of situations in both animals  and the human are most readily identified  during the stage of progression (Tamura et al., 1991; Navone et al., 1993; Tanaka et al., 1993; Donghi et al., 1993). In addition, abnormalities in the expression of the p16 tumor suppressor gene in melanomas are found fre- quently during the stage of progression but rarely in earlier stages (Reed et al., 1995). However, since karyotypic instability is unlikely to lead directly to point mutations in oncogenes and tu- mor suppressor genes, it is more likely that their appearance in malignant neoplasms reflects the selection of cells better suited to the growth environment  of the neoplasm, such cells already having these mutations. Thus, the stage of progression is a function not only of evolving karyo- typic instability but also of the selection of cells most suited for their aggressive replication and continued growth. Some cell and molecular mechanisms involved in the stage of progression are listed in Table 9.3. It should be noted that all of the characteristics in Table 9.3 can be directly associated with the evolving karyotypic instability, which may be considered the fundamental abnormality of this stage. Mechanisms associated with karyotypic instability are numerous and include disruption  of the mitotic apparatus,  alteration  in telomere function (Ledbetter,  1992; Blackburn, 1994), inhibition of topoisomerase function (Cortés et al., 1993), DNA hypomethy- lation (Smith, 1998), DNA and genetic recombination  (Chorazy, 1985; Murnane, 1990; Seng- stag, 1994), gene amplification  (Tlsty et al., 1993), gene conversion  (Taghian  and Nickoloff, 1997), and gene transposition (cf. Cheng and Loeb, 1993). As noted above, the relationship of alterations  in mismatch  repair with karyotypic  instability  is not entirely clear. Because of its unique role as “guardian of the genome,” abnormalities in the p53 tumor suppressor gene may also contribute to evolving karyotypic instability (Sood et al., 1997). As Shackney and Shankey (1997) have pointed out, p53 abnormalities become appreciable in many developing neoplasms just prior to the transition from preinvasive to invasive malignancy. Thus, it appears that there ar many pathways to the development of karyotypic instability in neoplastic cells in the stage of progression.

Mutational  macrolesions  (Table 7.6) resulting  from chromosomal  translocations,  dele- tions, recombinations, and other karyotypic changes have been discussed in Chapter 6 in relation to the formation of fusion genes. Other examples of gene rearrangement  occurring during the stage of progression  may be seen in experimental  and human myelomas, in which rearrange- ment of immunoglobulin  genes mimics in part the normal rearrangement  occurring  in these genes during the maturation of antibody-producing  plasma cells (Chapter 16). The small onco- genic DNA viruses whose genetic material is directly incorporated into that of the host cell may transform normal cells into neoplastic cells exhibiting the biological and molecular characteris- tics of progression (Rapp and Westmoreland, 1976). The T antigen, the product of the oncogene in such viruses, induces a large variety of chromosomal aberrations in transformed cells and may induce recombinational events in transformed and infected cells as well (cf. Fanning and Knip- pers, 1992). Some defective oncogenic RNA viruses lacking a specific transforming v-onc gene (Chapter 4) induce neoplasms in vivo only after a long latent period. Hayward et al. (1981) were among the first to demonstrate  that—at least in specific cases—the delay is related to the re- quirement for the insertion of the DNA of the virus in an appropriate position in relation to the proto-oncogene,  c-myc. Since that time, neoplasia resulting from retroviral integration into re- gions controlling the expression of specific cellular genes, termed insertional mutagenesis, has been described in a number of instances (Gray, 1991). Furthermore, Noori-Daloii et al. (1981) demonstrated the occurrence of gene amplification as well as structural alterations of the chro- mosomal region in which the insertion occurs. Other examples of such insertion occurring dur- ing the stage of progression have also been described (Breuer et al., 1989; Lazo and Tsichlis,1988). Such findings further indicate that the stage of progression may occur very early in the natural history of many virus-induced neoplasms.

Gene amplification is another characteristic of the stage of tumor progression that may be directly related to karyotypic instability (Tlsty, 1996). With a number of drugs—including meth- otrexate (Sharma and Schimke,  1994), nitrogen mustards  (Lewis et al., 1988), and etoposide (Campain et al., 1995)—amplification  of the target gene(s) of the drug occurs, with resulting drug resistance due to the marked increase in the amount of gene product. As a result of this induced gene amplification, the neoplastic cell may become quite resistant to the effects of the drug (cf. Chapter 18). Gene amplification may also be induced by carcinogenic agents such as the tumor promoter tetradecanoyl phorbol acetate (Chapter 7), which is capable of stimulating amplifications of several genes in cultured cells, including folate reductase (Varshavsky, 1981), metallothionein  I (Hayashi  et al., 1983), and SV40 DNA in the host genome of transformed hamster embryo cells (Lavi, 1981).

The instability of the genome of the neoplastic cell in the stage of progression makes plau- sible an extension of the “promoter insertion theory” (Chapter 4) as a potential mechanism for many of the characteristics of progression, especially those related to growth and gene expres- sion in this stage of development of neoplasms of viral, chemical, and physical origin. Further- more, aneuploid cells, including those in the stage of progression, may be more likely to be able to incorporate exogenous DNA into their genome than are normal diploid cells (e.g., Coonrod et al., 1997). Numerous examples of “transfection” of genes into aneuploid cells with subsequent stable expression of genes in a small number of progeny have been described (cf. Graf, 1982). During the recent past, the transfection of genes isolated from neoplastic cells into appropriate recipient cells has been utilized as a method for identifying cellular oncogenes or transforming genes (cf. Cooper, 1982). Initially, studies by several laboratories  identified a mutated Ha-ras proto-oncogene  as the transforming  gene occurring  in both human  and animal  neoplasms (Chang et al., 1982; Parada et al., 1982). Later studies demonstrated that specific base changes in the normal Ha-ras proto-oncogene resulted in this transformation (Tabin et al., 1982). Since then a number of such transforming genes, not all of which are derivatives of proto-oncogenes, have been described (Table 9.4). Land et al. (1983) demonstrated  that transfection  of two different “transforming genes” into third-passage rat embryo fibroblasts resulted in the neoplastic trans- formation. Transfection of the genes separately failed to induce such transformation. The anal- ogy between this phenomenon  and that of the stages of promotion and progression  in vivo is apparent. However, several studies have demonstrated that the transfection process itself can in- duce chromosome rearrangements and other types of mutations and epigenetic changes in recip- ient cells, leading to an induction of genomic instability in murine cells (Bardwell, 1989; Gilbert and Harris, 1988; Denko et al., 1994). Furthermore,  transfection  of a normal proto-oncogene into neoplastic cells in vitro enhanced karyotypic instability, so that the integration of the trans- fected gene occurred  predominantly  in aberrant chromosomes  (de Vries et al., 1993). Thus, while the transfection of specific genes has many potential uses, the interpretation of the “trans- formation” of cells in culture and possibly also in vivo may be somewhat difficult in view of these latter experiments.

The continuing evolution of the stage of progression  may also be related to irreversible changes in genetic expression. As noted above, alteration in methylation of the genome in neo- plastic cells can occur as an early event in neoplastic development, possibly even preceding the stage of progression, and may continue during the stage of progression, resulting in altered gene expression (Smith, 1998; Makos et al., 1993). As noted in Table 9.3, such mechanisms, in addi- tion to karyotypic instability, can lead to alterations in major groups of genes such as (1) those expressed during fetal life but not normally in the adult (Chapter 15), (2) repression and/or hy- perexpression of genes involved in histocompatibility  (Chapter 19), and (3) the ectopic produc- tion of hormones  by neoplasms  derived from cells that normally  do not express such genes (Chapter 18). In addition, the expression of genes important in DNA synthesis, the cell cycle, and apoptosis may also be abnormal in neoplastic cells in the stage of progression,  including cyclins (Imoto et al., 1997; Wani et al., 1997), telomerase (Shay and Wright, 1996), and the bcl-2 gene (Chapter 6) (Bronner et al., 1995). Hyperexpression of the c-myc proto-oncogene report- edly is an example of the altered expression of a gene during the stage of progression, primarily as a result of the amplification of the proto-oncogene (Garte, 1993). Drug resistance in neoplasia

Table 9.4 Mutations in Genes Whose Transfection Leads to Cell Transformation

has also been directly associated with karyotypic instability, first by Terzi (1974) and more re- cently by Duesberg et al. (2001), who observed that a major mechanism of drug-resistant muta- tions in cell lines was characterized by and probably the result of karyotypic instability. More recently, Schnipper et al. (1989) noted that, in an artificial system dependent on drug resistance, the frequency of resistance increased with duration of exposure to the clastogenic large T anti- gen. Studies have also demonstrated that the expression of a gene can be affected by its location in the chromosome, as when a gene is moved close to a heterochromatic region. This is termed the position effect (cf. Pardue, 1991). Finally, in a recent study, Li et al. (1997), investigating a system of chemical transformation of Chinese hamster cells (Chapter 14) in culture, noted that aneuploidy correlated completely with the induction of cell transformation  and presumed neo- plasia in this system.

On the basis of our knowledge of tumor progression and its characteristics, one may thus propose the following definition of this stage in the development  of neoplasia: progression  is that stage of neoplastic development  characterized  by the irreversible  evolution of karyotypic instability, which results directly in mutational macrolesions,  irreversible  changes in gene ex- pression, and the selection of neoplastic cells with genotypes/phenotypes  optimal for growth in the immediate environment. These characteristics  are further reflected by an increased growth rate, increased invasiveness,  successful metastatic growth, and alterations in biochemical and morphological characteristics of the neoplasm.

The latter part of the definition distinguishes the phase of progression from that of promo- tion, in which such major genetic changes have not been demonstrated. We have already noted (Chapter 6) gene rearrangements that occur during neoplastic development, many of these rear- rangements being somewhat specific for individual types of neoplasms (Table 6.8). By the above definition, these neoplasms are thus in the stage of progression. Other examples of gene rear- rangement occurring during the stage of progression are in experimental and human myelomas, in which rearrangement of immunoglobulin genes mimics in part the normal arrangement occur- ring in these genes during the maturation of antibody-producing plasma cells (Chapter 19).

The effect of the addition of exogenous genetic material, together with all of the apparatus needed for its expression, may be seen in virus-induced  neoplasms, especially those resulting from RNA viruses. When the virus is nondefective, as with the Rous sarcoma virus, a stage of promotion  cannot readily be discerned,  since tumor production  and growth are so rapid (Ha- nafusa, 1975). Similarly, many oncogenic DNA viruses, especially those whose genetic material is directly incorporated into that of the host cell, transform normal cells rapidly into neoplastic cells, which then exhibit many of the biological characteristics of progression (Rapp and West- moreland, 1976). On the other hand, certain defective oncogenic RNA viruses, especially those lacking a transforming v-onc gene (Chapter 4), induce neoplasms only after a long latent period. In several instances, this delay is related to the requirement for the insertion of the DNA of the virus in an appropriate position in relation to proto-oncogenes  (Chapter 4). Other DNA viruses that are clastogenic in human tissues include the hepatitis B virus (Simon et al., 1991), the hu- man papillomavirus (Hashida and Yasumoto, 1991), the cytomegalovirus  (Sakizli et al., 1981), and human polyomaviruses (Lazutka et al., 1996). Such oncogenic viruses that induce clastoge- nesis may be considered as progressor agents themselves.

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