Molecular Biology of Chemotherapy and Resistance
Adrienne C. Scheck, PhD
Division of Neurology, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix, Arizona
The recurrence of human gliomas several months after treatment suggests that intrinsic, rather than acquired, resistance underlies the failure of therapy. Therapy reduces cellular heterogeneity, allowing only resistant cells to survive. Mechanisms of therapy resistance must be understood before new treatment modalities can be developed. Consequently, this article reviews resistance to therapy-induced cell death after treatment with a variety of chemotherapeutic alkylating agents and topoisomerase inhibitors, along with the role of gene therapy in designing more efficacious< therapies.
Key Words : apoptosis, BCNU, cisplatin, chemotherapy, gene therapy, gliomas, resistance, tumors
Human malignant glioma is an almost uniformly fatal disease. Despite decades of research, life expectancy after its diagnosis has not substantially changed. Surgical resection of the primary tumor followed by irradiation and chemotherapy is a palliative measure, and the tumor typically recurs. The recurrent tumor is often detected 3 or 4 months after treatment, suggesting that intrinsic rather than acquired resistance is responsible for the failure of therapy. Thus, within the heterogeneous population of cells that comprises the primary tumor, a subpopulation of cells is intrinsically resistant to therapy and capable of surviving and growing in the tumor bed after therapy.57, 91 If the mechanisms of therapy resistance could be determined, the way would be paved for the development and application of additional adjuvant therapies and treatment modalities.
The Cell Cycle, Apoptosis, and Resistance to Therapy
Considerable interest has been focused on the cell cycle and the role of the tumor suppressor p53 in investigations of resistance to therapy. During normal cell growth and division, the cell traverses through the cell cycle in an ordered manner (Fig. 1). At the boundaries of G1®S and G2®M, however, there are “checkpoints” where the cell cycle can stop if damage to the deoxyribonucleic acid (DNA) is detected. At these times the cell may attempt to repair the damage, and a decision is made either to continue the cell cycle or to initiate a cascade of events that leads to cell death through a process known as programmed cell death or apoptosis. A complete discussion of apoptosis is outside the scope of this review; however, the importance of this process in the cellular response to therapy warrants a brief discussion.
Apoptosis is characterized by membrane blebbing with intact membranes, cell shrinkage leading to the formation of apoptotic bodies, chromatin condensation, and nonrandom DNA fragmentation that causes DNA laddering. The “balance” of a number of genes, including the pro-apoptotic genes bax, bak and bad as well as the apoptosis suppressors bcl-2 and bclxL, affects cell survival or apoptosis directly. In many systems, apoptosis is initiated rapidly after cytotoxic therapy, and the process is often dose dependent.
Resistance to therapy-induced apoptosis may be a mechanism of resistance in a variety of tumor systems and is likely to be linked to alterations in the cell cycle at the checkpoints as a result of therapy-induced DNA damage. Thus, the regulation of the cell cycle at these checkpoints is crucial to how cells respond to therapy. Correspondingly, the roles of TP53 and related genes [e.g., p21WAF1/CIP1and murine double minute 2 oncogene (MDM2)] in this response have received considerable attention.38
In some cells with a wild type p53 phenotype, p53 is induced by damage to the DNA. Its induction causes the arrest of G1, which allows time for the DNA to be repaired before its replication. If the DNA is repaired, the cell goes through a normal cell cycle. If the DNA cannot be repaired, cells may undergo apoptosis,29 presumably through the up-regulation of bax and the down-regulation of bcl-2 expression. The cell cycle of cells with a mutated or missing TP53 gene may not be arrested.47,66 Consequently, the damaged DNA is replicated and may contribute to tumor progression.
Alterations in genes that modulate the activity of p53 can mimic the p53-deficient phenotype, even in the presence of a normal TP53 gene. For example, the p21WAF1/CIP1 gene is induced by p53 and may be responsible for the p53-dependent checkpoint at G1. Colorectal cancer cells deficient in p21 do not undergo p53-dependent arrest of G1 after DNA damage,112 either in vivo or in vitro.24Additional studies have demonstrated that p21-deficient cells undergo apoptosis after DNA damage.113 In some systems, however, p53 is required for the initiation of apoptosis.56,66 DNA damage caused by radiation or chemotherapeutic agents such as cis-diaminedichloroplatinum(II) (DDP, cisplatin) can increase the levels of messenger ribonucleic acid (mRNA) encoding MDM2.80Furthermore, the accumulation of p53 protein increases in cisplatin-resistant ovarian tumor cell lines.12,29 Adenovirus- or transfection-mediated transfer of wild-type p53 into cells increases their sensitivity to cisplatin.12,31 In contrast, astrocytes from p53 knockout mice with wild-type p53 were more resistant to 1,3-bis-(2-chlorethyl)-1-nitrosourea (BCNU, carmustine) than those from p53-null mice.71Thus, the action or actions of TP53 and related genes with respect to arrest of the cell cycle, apoptosis, and/or resistance to therapy vary in different cell types and depend on the genetic background of the cells (e.g., other oncogenes, growth factors). An understanding of the full impact of these genes on resistance to therapy is likely to require additional insights into the inter-relationships between these genes and other modulators of cell growth.
The chemotherapeutic agent most commonly used in the treatment of gliomas is BCNU (Fig. 2), a lipophilic, bifunctional, alkylating agent that crosses the blood-brain barrier. This feature facilitates its use in brain tumor therapy. In an aqueous environment, BCNU acts as both an alkylating and a carbamoylating agent capable of damaging both nucleic acids and proteins. The major cytotoxic event related to BCNU treatment is alkylation of guanine at the O6 position (Fig. 3). If unrepaired, this damage leads to misreading of the DNA code, to covalent cross-linking of the DNA, or both.
In addition to BCNU, similar agents such as cisplatin and its analogue cis-diamine 1,1-cyclobutane dicarboxylatoplatinum II (carboplatin) are used in the treatment of gliomas (Fig. 2). Like BCNU, these drugs also form intra- and interstrand cross-links in DNA and protein. Streptozotocin is an antibiotic isolated from Streptomyces achromogenes . Its principal mechanism of action is thought to be methylation of DNA; it does not alkylate DNA like BCNU.
A number of genes may be involved in resistance to BCNU and other alkylating agents. For example, the gene for methylguanine methyltransferase (MGMT) is located on chromosome 10,83 and MGMT is active in the repair of nitrosourea-induced DNA damage. This protein reacts with the DNA such that the alkyl group is transferred from the DNA molecule to a cysteine acceptor site. The acceptor site cannot be regenerated. Thus, MGMT is known as a suicide enzyme—each protein molecule can remove one alkyl group from the DNA and is then irreversibly inactivated. Typically, cells with little or no methyltransferase activity (often termed mer-) are more sensitive to alkylating agents than are cells that express this activity (termed mer+). MGMT is expressed in human brain tumors30,102,103 and is elevated in some but not all BCNU-resistant glioma cell lines.76,86 Furthermore, the expression of this protein is heterogenous within cells in the same tumor,52 and alkyltransferase immunoreactivity may be correlated with the survival of patients treated with BCNU.7
The gene encoding MGMT has been cloned.40,108,111 Furthermore, the expression of MGMT with concomitant alterations in sensitivity to BCNU can be modulated through transfection experiments (to increase MGMT activity).23 Compounds such as O6-benzylguanine and streptozotocin inactivate MGMT and increase the cytotoxicity of BCNU in resistant cells.2,20,34-36,58-60,78 In addition to MGMT-mediated repair, DNA mismatch repair22 and nucleotide excision repair14,15 (Scheck and Shah, unpublished data) may be involved in resistance to BCNU. Thus, the role of DNA repair in resistance to alkylating agents is likely to be multifaceted, and intervention may therefore require multiple approaches.
Cells may also be resistant to alkylating agents through detoxification via conjugation with glutathione (GSH). GSH is an important component of drug resistance in gliomas, and the overexpression of enzymes involved in the GSH redox cycle [e.g., glutathione-S-transferases (GSTs) and gammaglutamyl transpeptidase (GGT)] has been implicated in resistance to alkylating agents. The prevention of DNA damage by the “scavenging” of free radicals by sulfhydryl compounds such as GSH may also contribute to intrinsic radioresistance.9,18,63 Reduction of the amount of GSH with drugs such as buthionine sulfoximine increases a cell’s sensitivity to both radiation and chemotherapy.73 Three classes of GST isoenzymes (a, m, p) have been implicated in resistance to alkylating agents in a number of human and animal cells, including gliomas.17,51,81,101,105,109,114 In glioma cell lines, the expression of GST-p also correlates with the degree of resistance to BCNU.3
Another enzyme in the GSH cycle, GGT, is involved in the biosynthesis of GSH. GGT is mapped to chromosome 22q11.2 and may play a role in the resistance of some human malignant glioma cell lines106 and other tumor cells53 to nitrosoureas. Furthermore, the multidrug resistance-associated protein (MRP) has been implicated in the GST-mediated detoxification by acting as an efflux pump for GSH-conjugated molecules.67,118 However, the gene copy number and/or expression of these genes is not elevated in all resistant cells (Tables 1 and 2). The multidrug resistance gene (MDR) locus is mapped to chromosome 7 and encodes a highly homologous family of proteins that also act as energy-dependent drug efflux pumps.25 The presence of p-glycoprotein (the MDR gene product) has been found only in the endothelial cells of normal brain,19 and the expression of protein, mRNA, or both varies in some, but not all, gliomas and/or glioma cell lines.6,41,61,68 We, too, have found only a small increase in the gene copy number and mRNA expression of the MDR gene in only a few glioma cell lines (Table 1). Overexpression of this protein has not been demonstrated in BCNU-resistant gliomas,68 and cells with an MDR phenotype are often, but not always, sensitive to BCNU and cisplatin.44,45
A number of resistance mechanisms have been described for cisplatin and other platinum-containing drugs, mostly from work with ovarian cancer cells. A number of genes play roles similar to those involved in resistance to BCNU. GSH and genes of the GSH cycle may be elevated in resistant cells,4,39,48,51,62 leading to drug detoxification. Reduction of GSH by transfection of a GST-p antisense construct also reduces the resistance of a colon cancer cell line to cisplatin.4 Furthermore, the expression or activity of genes involved in DNA repair, such as those of the thymidylate synthase cycle84 and DNA polymerase b,49 increases in some cells.
Recently, attention has shifted to mechanisms of DNA repair, including gene interactions and mismatch repair.1,13,21,26,27,69,79,104The isolation of genetic suppressor elements and changes in sensitivity to cisplatin when these elements are introduced into resistant cells suggest a role for p53 in resistance to cisplatin.33 Furthermore, mutations of TP53 are found in cells resistant to cisplatin.75
Resistance to Alkylating Agents
Comparative genomic hybridization studies of two human ovarian cancer cell lines and derivatives selected for resistance to cisplatin have shown a gain of material from chromosomes 2q, 4, 6q and 8q as well as a loss of material from chromosomes X, 2p, 7p, 11p, and 13.115 In contrast, somatic cell hybridization studies using one of the same ovarian carcinoma cell lines have demonstrated that resistance segregated with chromosomes 11 and 16. Whether a gene or genes from these chromosomes are actively involved in resistance to cisplatin remains to be seen. Such studies, however, provide a background on which to base further molecular studies. This is the strategy used in our laboratory to analyze BCNU resistance in gliomas.
Although many brain tumor cells are sensitive to damage by BCNU, the karyotypic heterogeneity in primary malignant gliomas100 and differential sensitivity of individual cell types to BCNU are substantial.117 Bradford et al.10 replicated these findings and also showed differences in the drug sensitivity of clones derived from a murine astrocytoma. At the level of individual genes,86,88,89 human glioma cell lines show a similar heterogeneity.
When cells from the primary tumor are grown in the presence of clinically achievable concentrations of BCNU (blood plasma levels, ~10 mg/ml) or are cloned from a colony-forming assay after a single exposure to BCNU,97 a BCNU-resistant subpopulation of cells is selected in vitro that is near-diploid.97 These cells carry a specific nonrandom karyotypic deviation that includes overrepresentation of fragments or whole chromosomes 7 and 22.87,92 Approximately 30% of the tumors studied to date have this subpopulation at the time of the primary resection. When treated with radiation and BCNU, these patients develop recurrent tumors in which this same cell type (near-diploid with overrepresentation of some or all of chromosomes 7 and 22) is the dominant population.99 Our molecular analyses of drug-resistant cells with overrepresentation of chromosomes 7 and 22 have not consistently detected large differences in the copy number or expression of genes known to be involved in BCNU resistance (Tables 1 and 2).
A new class of GST—GST-q—has been localized to chromosome 22. There are at least two isoenzymes in this family,43 one of which is polymorphic and absent from approximately 38% of the population.74
GST-q appears to play a role in detoxifying carcinogenic chemicals such as halomethanes;74 however, a role for this enzyme in resistance to chemotherapy has not yet been described.
To determine if this isoenzyme is involved in resistance to BCNU, we used a cell line that lacked MGMT activity. BCNU-resistant clones were isolated from this cell line, and the expression of GST isoenzymes was determined (Table 2). GST-q did not appear to play a role in BCNU resistance in these cells. Other GST isoenzymes were overexpressed in some, but not all, resistant clones (Table 2). Furthermore, genes known to be involved in BCNU resistance (mainly MGMT and the GSTs) are not mapped to chromosomes 7 and 22 (except GST-q). This finding suggests that the overrepresentation of chromosomes 7 and 22 involves other genes related to survival, growth, or both after drug therapy.
Nor is drug resistance a simple matter of increased gene copy number resulting from karyotypic abnormalities. MGMT is mapped to chromosome 10, a chromosome frequently underrepresented in gliomas. Many of our cell lines, however, contain a normal or increased copy number and expression of the MGMT gene. So, perhaps, while the chromosome is lost, this gene (which is probably required for survival in some cells after drug treatment) is functionally retained somewhere in the genome. This possibility underscores the importance of gene expression studies. We have also correlated overexpression of the GSTs with BCNU resistance in some, but not all, of our BCNU-resistant tumor cell lines (Table 2).
The lack of consistent correlation between the expression of MGMT and/or GSTs and drug resistance has a number of possible explanations. It is likely that gliomas use more than one mechanism to survive adjuvant therapies. Our work and most of that reported in the literature have demonstrated that both MGMT and a number of GST isoforms are involved in BCNU resistance. The heterogeneity that is prevalent in these tumors extends to drug-resistance mechanisms. Although some tumors appear to use GST(s) or MGMT, some appear to use both and some do not appear to use either. Liang55 reached a similar conclusion when he was unable to demonstrate increases in the expression of these same genes after hypoxia-induced increases in BCNU- and cisplatin-resistance in human glioma cell lines.
Topoisomerase II Inhibitors: Etoposide and Teniposide
The topoisomerases play a crucial role in the normal processes of DNA replication and transcription by facilitating DNA breakage that allows DNA supercoils to relax. Topoisomerase II specifically cleaves double-stranded DNA and bridges the break while another piece of double-stranded DNA passes through. Etoposide (VP-16) and teniposide (VM-26) are topoisomerase II inhibitors that exert their effect by stabilizing the cleavable complex of the DNA-topoisomerase II. These drugs do not directly interact with DNA, suggesting a mechanism of action (and mechanism(s) of resistance) specifically related to the topoisomerase II enzyme.
Resistance to these drugs could result from diminished amounts of topoisomerase II. In general, quiescent cells have little detectable topoisomerase II. In contrast, proliferating cells have many copies of these molecules, suggesting that faster-growing tumors should be more sensitive to these therapies than tumors that grow more slowly. However, topoisomerase II is down-regulated in some resistant tumors. Considerable evidence indicates that alterations in one or more domains of the topoisomerase II molecule itself can create resistance. Molecules with increased resistance to a cleavable-complex formation, altered catalytic activity, an altered ATP-binding domain, and/or an altered drug-binding domain can affect the cells’ sensitivity to VP-16 and VM-26.37 Furthermore, although the cleavable complex is relatively short lived, the presence of these complexes leads to permanent DNA damage. This damage is likely mediated by the DNA synthesis machinery itself because inhibitors of DNA synthesis can (at least partially) restore resistance.
A number of approaches to gene therapy have been used in attempts to design more efficacious therapies and are reviewed elsewhere.82 Briefly, however, these therapies are composed of two main “parts”: a vector-delivery system and a therapeutic agent. The vector-delivery system can be a genetically engineered virus, plasmid, or cell that is somehow delivered or targeted to the tumor. Many of these proposed therapies have shown promise in rodent models. Their efficacy in humans, however, is under intense scrutiny and has yet to be established.
Perhaps the best known therapy is the HSV-tk/ganciclovir system (Fig. 4). In this strategy, the thymidine kinase (tk) gene from theHerpes simplex virus (HSV) is delivered to the tumor cell using a virus vector such as a genetically altered adeno- or retrovirus. The patient is then treated with ganciclovir. The HSV-tk phosphorylates the ganciclovir to the monophosphate form. Cellular phosphorylases can then phosphorylate it to the triphosphate form. In this form it is incorporated into cellular DNA, resulting in chain termination causing cell death. Despite its successful use in rodent models, this therapy has met with limited success in humans.
Ongoing trials with this therapy, however, are providing information about the cytotoxic effects of this system on cells with and without the virus construct. The so-called “bystander effect” appears to mediate cytotoxicity in cells that have not received the HSV-tk gene. The mechanisms underlying this effect are unknown, but they may be related to the transfer of metabolic products through gap junctions, phagocytosis of dead or dying cells, or changes in the host’s immune system. Regardless of its source, the bystander effect is an important part of gene therapy.
The transfer of drug-resistant genes (Fig. 5) such as MGMT or the multidrug resistant gene (MDR-1) is also being studied in an attempt to “protect” the host from chemotherapeutic side effects, thereby allowing the use of higher doses of toxic therapeutic agents. Thus, the addition of MGMT to hematopoietic cells helps to protect these cells from the unwanted side effects of treatment with BCNU. Genes that modulate the host’s immune system are also being transduced in an attempt to cause the host to mount an immune response against the tumor.
In addition to therapies directly involving chemotherapeutic agents, trials in which oncogene expression is reduced or blocked are ongoing. In these systems, a vector is used to deliver an antisense molecule (Fig. 6) or a ribozyme (Fig. 7) specific for a particular oncogene. Antisense molecules are nucleic acid fragments whose sequence is complementary to the gene of interest. When added to the cell, it base-pairs with the mRNA or DNA and inhibits transcription, splicing, transport, and/or translation of the message. Ribozymes are catalytic RNA molecules designed to cut a specific sequence in the mRNA molecule. In both cases, the expression of the targeted oncogene is reduced.
Tumor suppressor genes are another avenue of investigation. Some gene therapies, for example, are designed to reintroduce wild type tumor suppressor genes (such as TP53) to cells lacking its normal expression. Also being tested are strategies that block or inhibit a tumor’s ability to induce angiogenesis, thus preventing the tumor from enhancing its blood supply.
Investigations of new therapies are ongoing.16 More than 200 new agents are being tested in clinical trials of all forms of cancer. Despite the enormous increase in gene therapy trials during the last few years, a major hurdle must be overcome before these therapies can be used to their full potential. The delivery systems used to deliver these genes are still far from optimal. Vectors and delivery systems must be designed to deliver the genes safely to the appropriate cells and to express them in a therapeutically useful way.
Molecular Biology and the Future of Brain Tumor Therapy
The presence of intrinsically resistant cells in the absence of the overexpression of a particular gene or genes suggests the presence of other mechanism(s) of resistance. A possible explanation for such resistance may relate to gene interactions that occur in response to therapy. One such example is the postulated connection between immediate-early regulatory genes and resistance to therapy. In particular, work in a number of systems has demonstrated a link between the expression of c-fos and the induction of genes involved in DNA repair and/or drug resistance.
The expression of c-fos can be altered by treatment with alkylating agents. These changes have been demonstrated in two cancer cell lines. In a colon carcinoma line, the steady-state levels of c-myc and c-fos transcripts have been increased.32 In a cisplatin-resistant ovarian carcinoma line, the expression of c-fos was increased along with c-H-ras, thymidylate synthetase, and DNA polymerase b.46Suppressing the induction of c-fos reduces these cells’ resistance to drugs.85 The induction of GST is mediated by regulatory elements that are activated by fos/jun heterodimeric complexes (AP-1).8 Therefore, the induction of c-fos by a growth factor or other mechanism may stimulate the production of genes associated with DNA repair, resulting in a more resistant phenotype. In addition to gene interactions that alter transcriptional activation, genes involved in the control of the cell cycle may play a role in DNA repair and resistance to therapy, as described above.
Thus, the role of many genes and gene interactions in the resistance to therapy is only now being realized. Likely, other as yet unidentified genes are also involved. Our work is aimed at identifying these genes by comparing gene expression in drug-sensitive cells with that of drug-resistant cells using a technique known as differential mRNA display (Fig. 8).
This technique is based on reverse transcription-polymerase chain reaction technology. Instead of designing primers to a specific gene, however, random primers are used so that all mRNAs in the cell are amplified. These RNAs are then separated and visualized on a polyacrylamide gel, and the resulting patterns (representing gene fragments) are compared (Fig. 9). Differences in the pattern may be indicative of differential gene expression. These fragments are then cloned and sequenced to determine their identity.
We have used this strategy to identify genes that may be involved in resistance to BCNU therapy. The presence of differentially expressed RNAs in BCNU-sensitive (untreated) and BCNU-resistant (5 µg/ml) cells was demonstrated,70,90 and the fragments were reamplified, cloned, and sequenced. We have identified one overexpressed gene as the transforming growth factor b1 binding protein (LTBP-1).
The potential role of LTBP-1 in BCNU resistance is completely unknown and has not been described previously. LTBP-1 is a component of the latent transforming growth factor b (TGF-b) complex that is required for activation of TGF-b1. The gene encoding this protein has been mapped to 19q13.1, and additional related proteins have recently been identified.28,64,107,110 The precise role of these proteins in the activation of latent TGF-b1 has not been determined, but the overexpression of this gene in BCNU-resistant HFA cells is intriguing. TGF-b1 interacts with platelet-derived growth factor (PDGF) and/or its receptors,5,11,50,54,72,116 and it may be involved in apoptosis65 or in resistance to the therapeutic agent N-phosphonacetyl-L-aspartate (PALA).42 Furthermore, overexpression of this gene could have an indirect effect on the regulation of the cell cycle, which may also ultimately affect the cell’s ability to survive therapy.
Given that some BCNU-resistant cells overexpress PDGF,87 this finding is particularly intriguing. The roles of this gene and other as yet unidentified genes found by our differential mRNA display experiments in BCNU resistance are being investigated. Through these studies we hope to identify novel mechanisms of therapy resistance.
The resistance mechanisms in tumors cannot be analyzed without understanding the biology of the system. Most techniques are “population” techniques—that is, the results represent an average of many cells. Even data obtained from cultured cells can be influenced by the dynamics of the culture and the selection pressure(s) placed on cells by their environment. The reported variation in terms of resistance mechanisms in gliomas and other neoplastic systems reported in primary and recurrent human gliomas underscores this possibility.92-96,98 Cells seen as a subpopulation in the primary tumor become the dominant population in tumors that recur after treatment. Similar results have been obtained by Phillips et al.,77 who found that a subpopulation of cells in a nude mouse xenograft system can expand selectively after treatment with BCNU.
Despite the caveats inherent in analyzing these heterogeneous tumor systems, it is clear that the mechanisms of resistance used by cancer cells to survive treatment with these (and related) compounds are varied. It is equally clear that therapy reduces this heterogeneity, allowing only resistant cells to survive. As our understanding of the biology of gliomas and the body’s response to therapy increases so will the opportunity to design improved therapeutic modalities, which will likely assume many forms. Not only will we need to devise new therapies, but it may be possible to alter the efficacy of current therapies by modulating the expression of genes involved in therapy resistance. Consequently, scientists are looking for novel genes and studying the regulation of DNA repair in response to damage induced by therapy; the regulation of genes that alter the cell cycle and cell growth; and the interplay among cell growth, cell death, and resistance to therapy.
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