Some 25 years ago, Terzi (1974) pointed out that drug-resistant mutants of some cell lines were characterized by karyotypic instability, a high reversion frequency, and low plating efficiency. Subsequent studies have supported these initial observations in demonstrating alteration of re- sponse to drug therapy in cells by induced DNA rearrangements (Schnipper et al., 1989), hy- poxia-inducing genetic instability in neoplastic cells (Teicher, 1994), and the importance of tumor heterogeneity resulting from karyotypic instability in the response of neoplasms to spe- cific drugs (Simpson-Herren et al., 1988). Another closely related mechanism of drug resistance was initially described by Schimke, who demonstrated that cells resistant to the antifolate meth- otrexate exhibited a dramatic amplification of the gene to which the drug dihydrofolate reduc- tase was targeted (Schimke, 1984). Subsequent to those studies, a number of examples of drug- induced gene amplification in karyotypically unstable cells have been reported (Table 20.7). In addition to the examples listed in the table, examples of induced gene amplification of topo- isomerase II by etoposide in human melanoma cell lines (Campain et al., 1995) and the amplifi- cation of metallothionein genes by metals (Gick and McCarty, 1982) and potentially by alkylating agents (Kelley et al., 1988) have been reported.
Figure 20.10 A schematic representation of some molecular mechanisms of resistance to chemothera- peutic agents within a neoplastic cell. (After Harrison, 1995, with permission of the author and publisher.)
In addition to induced gene amplification altering the DNA target, a variety of mecha- nisms resulting in resistance to the topoisomerase inhibitors have also been described, only some of which involve these mechanisms (cf. Skovsgaard et al., 1994). Alteration of the DNA target by hypermethylation induced by chemotherapeutic agents, resulting in altered responses to such drugs, has also been reported (Nyce et al., 1993).
Pharmacogenetics of Drug Resistance
A number of chemotherapeutic agents require “bioactivation” to produce pharmacologically ac- tive, cytotoxic species (cf. Sladek, 1987). Both phase I and phase II enzymes (Chapter 3) have been identified as responsible both for differential sensitivity of individuals to chemotherapeutic agents as well as the response of neoplastic cells themselves to specific agents (cf. Iyer and Ratain, 1998; Graham et al., 1991; Chang et al., 1994). Cyclophosphamide and ifosfamide are alkylating agents that require bioactivation by phase I enzymes, although such bioactivation oc-
Figure 20.11 Proposed mechanism of action of P-glycoprotein as a “hydrophobic vacuum cleaner.” As noted in the model, the multidrug transporter may remove drugs directly from the plasma membrane or from the cytoplasm through a single transport channel, as illustrated. (After Gottesman, 1993, with permis- sion of the author and publisher.)
curs predominantly in the liver. While it is possible that some other chemotherapeutic agents requiring phase I metabolic activation may do so at very reduced rates in neoplastic cells, thus leading to drug resistance, it appears that the changes in the phase I enzymes, especially those involved in glutathione metabolism, are probably the most important in this potential mecha- nism of drug resistance (cf. Graham et al., 1991; cf. Iyer and Ratain, 1998).
DNA Repair Mechanisms and Cellular Resistance to Chemotherapeutics
As noted in Chapter 3, the cell contains a variety of mechanisms responsible for the repair of damaged DNA. Closely related to this fact is also the finding that a large number of neoplasms exhibit mutations in genes whose products are involved in the recovery of cells from DNA dam- age, such as the p53 gene. In examining the various types of DNA repair noted in Table 3.4, there is ample evidence that the O6-alkylguanine-DNA alkyltransferase (AGAT) enzyme, when expressed at high levels, enhances the resistance of a cell, normal or neoplastic, to alkylating agents that produce O6-alkylguanine. Transgenic mice expressing increased levels of AGAT
Table 20.7 Gene Amplification Induced by Chemotherapeutic Drugs
were found to be protected from alkylating agent treatment having a specificity for the tissues in which the AGAT transgene was expressed (Liu et al., 1996). The complexity of the mechanism of excisional DNA repair involving both short and long patches (Chapter 3) suggests that resis- tance mechanisms in this pathway would also be complex. At least some of the proteins of this pathway, namely, DNA polymerase β when overexpressed in neoplastic cells, result in a pheno- type resistant to the effects of drugs causing alkylation and/or single strand breaks. Increased levels of nucleotide excision repair of interstrand crosslinks have been reported in cells resistant to a variety of DNA-damaging agents (cf. Barret and Hill, 1998). In addition, the expression of ERCC1 and ERCC2, genes whose products are involved in excisional DNA repair, are found to be increased by two- to threefold in neoplasms exhibiting a clinical resistance to therapy with platinum compounds (Dabholkar et al., 1992). There is also significant evidence that mismatch repair deficiency may mediate the resistance of human neoplasms to specific chemotherapeutic agents (Brown, 1999). While in some instances the exact proteins involved in resistance to DNA damaging agents are not certain, the increased activity of at least one important protein involved in DNA repair, poly(ADP) ribose polymerase, has been reported in some examples of drug resis- tance to both alkylating agents and chemotherapeutic antibiotics (Urade et al., 1989; Chen et al.,1994). Evidence for the involvement of double-strand DNA break repair in resistance to chemo- therapeutics is rare, but there is a suggestion that decreased mismatch repair may contribute to the resistance of some chemotherapeutic drugs (cf. Barret and Hill, 1998). Thus, while this is an important area for potential drug resistance, the exact mechanisms involved in resistance of var- ious chemotherapeutics, especially alkylating agents involving DNA repair mechanisms, needs considerable further investigation.