Radiation and chemotherapy are two modalities used to treat GI cancers and improve survival rates. Anticancer drugs are targeted to a multitude of intracellular components disrupting normal cellular homeostasis and/or inducing cellular damage. Despite this diversity of intracellular targets, cytotoxic anticancer drugs converge to a common response, cell cycle arrest and the induction of apoptosis.71 Furthermore, it is evident that the same oncogenes and mutations in tumor suppressor genes that are implicated in the induction of cancers can also explain, in part, the resistance of certain tumors to these conventional treatment modalities. In this section we will discuss the role of the cell cycle and apoptotic machinery in the cytotoxic effects of and resistance to chemotherapeutic agents and radiation therapy.
The use of chemotherapy in the treatment of GI cancers is to target presumed micrometastasis and established macrometastasis, which are beyond the scope of surgical resection, to improve overall survival rates. Many chemotherapeutic agents with diverse mechanisms of action have been shown to induce apoptosis in a variety of GI tumors and cancer cell lines. Despite this diversity of chemotherapeutic action, the cytotoxic process which follows have been broken down into four stages which lead to the induction of apoptosis.149 In the first stage, the chemotherapeutic agent disrupts cellular homeostasis through a specific interaction with an intracellular target (i.e., RNA, DNA, or microtubules) resulting in its dysfunction. The second stage involves the recognition by the cell of homeo-static disruption (i.e., p53 response to DNA damage). In the third stage, the cell determines the severity of the insult, and makes the decision whether to repair the injury or proceed to apoptotic cell death. Finally, stage four is the induction of the apoptotic machinery leading to the morphological and biochemical features of this process. Although many chemothera-peutic agents are used to treat GI malignancies, the specific mode of action of each drug in relation to the induction of apoptosis is not well understood.71 The observation that antitumor drugs with disparate modes of action converge to induce cell death by apoptosis suggests that it is not the drug-induced lesion that causes apoptosis, but subsequent events such as disruption of growth.71 Indeed many of the current chemotherapeutic agents have a profound impact on cell cycle progression through cross-linking nucleic acids (alkylating agents), interfering with DNA and RNA synthesis (antimetabolites), and inhibiting mitosis through the binding of microtubules (vinca alkyloids). Each of these drugs has been shown to induce apoptotic cell death.71,156 In our laboratory, we have studied the effects of olomoucine and roscovitine, novel compounds designed to inhibit Cdks, on four human gastric cancer cell lines157 and five pancreatic cancer cell lines (unpublished data). We found that these compounds completely block Cdk2 and cdc2 activities and inhibited cellular proliferation with cell cycle arrest at the G2/M transition. Furthermore, roscovitine resulted in increased apoptosis by biochemical and morphological criteria in each of the pancreatic cancers studied. These data suggest that disruption of cell cycle events can act as a trigger to initiate the apoptotic cascade. Furthermore, these data show how 'designer' drugs aimed at the cell cycle machinery can be effective in inhibiting cancer cell proliferation and inducing cell death.
Radiation and chemotherapy are treatments used to decrease tumor burden. The primary impact of radiation on the cell is a result of DNA damage, which activates p53 for G1 cell cycle arrest to allow for DNA repair, or the induction of apoptosis. It is known that chemotherapeutic agents induce p53-dependent cell cycle arrest, and it has been suggested that anticancer agents induce p53-mediated apoptosis.71 Although this may explain, in part, the resistance seen in some cancers with mutated p53 gene, it provides no insight as to why such treatment modalities are used to successfully treat tumors. Recently, a hypothesis has emerged that implicates intact cell cycle checkpoints involving cell cycle arrest as critical mediators of the response to chemotherapy and radiation. The p21 gene is transcriptionally activated by p53, and is responsible for the G1 cell cycle arrest following DNA damage. It has been shown that cancer cells with a defective p21 response as a result of deletion or p53 mutation underwent apoptosis as a result of DNA damage or chemotherapeutic drug treatment.158 However, cancer cells with an intact p21 checkpoint, which is also found in normal human cells, entered a stable cell cycle arrest. Furthermore, it was demonstrated that loss of the p21 checkpoint resulted in the continuation of DNA synthesis without mitosis, resulting in polyploidy and apoptosis. Similar results were obtained in a recent in vivo study using xenografts which were established from isogenic colon cancer cell lines differing only in their p21 checkpoint status.159 This uncoupling of mitosis and S phase after DNA damage suggests a mechanism to account for the specificity of commonly used chemotherapeutic agents and ionizing radiation, and supports the investigation of novel compounds that induce checkpoint-dependent cell cycle arrest as anticancer drugs.
Resistance of cancer cells to the effects of chemotherapy and radiation treatment seems to have a common central theme, reduced sensitivity to the induction of apoptosis.149 Many human cancers express a mutated p53 tumor suppressor gene. Wild type p53 is a potent inducer of apoptosis after DNA damage. It has been shown that loss of p53 function results in drug and radiation resistance to apoptosis.160 Furthermore, increased expression of
Table 4.1 Challenges to gene therapy
I. Technical obstacles
1. Improvements in anatomic physical delivery systems for effective gene transfer to solid tumors
2. High-efficiency tumor-specific transduction producing a direct or indirect biologic effect against all tumor cells
3. Design of safe tumor-restricted replication effective vectors
4. Development of novel vectors capable of carrying large complex gene constructs
5. Cost-effective large scale production of vectors
B. Effector gene strategies
1. Development of tumor-specific strategies (e.g., tumor specific receptor mediated uptake, tumor-specific promoters)
2. Requirement for a range of effective antitumor gene constructs tailored to tumors of a particular molecular genetic profile (e.g., p53-deficient, DNA mismatch repair-deficient)
3. Inducible systems to control transgene expression
II. Potential or proven risks
1. Vector related (e.g., viremia, inflammation)
2. Vector DNA recombination, leading to production and release of replication-competent virus
3. Large volumes of cationic lipids
4. Deleterious effects of suicide gene on normal tissues (e.g., liver)
5. Prodrug toxicity (not a major problem with current drugs)
6. Transgene toxicity
B. Induction of neoplastic transformation
1. Insertional mutagenesis
2. Unknown effects of p53 overexpression in nontumor cells
C. Induced autoimmunity against 'self' antigens after successful tumor killing/immune modulation
Reproduced with permission (Zwacka RM, Dunlop MG, Hematology/Oncology Clinics of North America 1998; 12:599.
anti-apoptotic regulators (i.e., Bcl-2 and Bcl-xL), or decreased expression of pro-apoptotic regulators (i.e., Bax) correlate with resistance to radiation and chemotherapy in a variety of human cancers.161 It is evident that certain genetic mutations that confer cell survival advantages in tumorigenesis are also responsible for the resistance to treatment. Apoptosis-related genes and their mutations seem to provide a prognostic marker and predictor to chemoresponsiveness in many cancers.
As described in this review, aberrancies in cell cycle regulation and the apoptotic pathway are key events in the induction and progression of cancers. The abundance of research in these fields has provided information on the genetic mutations involved in cancer formation. Although cancer generally arises as a result of multiple genomic perturbations, the detailed knowledge of these events has opened new opportunities to develop novel therapeutic modalities, such as gene therapy. Gene therapy involves the introduction of genetic material into target tissue to achieve therapeutic benefit.162 One of the major strategies utilized in cancer gene therapy is the re-establishment of wild type tumor suppressor gene function.163,164 Of particular interest is the p53 tumor suppressor gene because of its frequent incidence of mutation in many GI cancers. As described above, wild type p53 is necessary for cell cycle arrest and the induction of apoptosis after genotoxic damage. Loss of this functional role results in uncontrolled proliferation and the progression of genetic mutations to daughter cells, and has also conferred resistance to standard chemotherapy. Therefore, the re-establishment of wild type p53 gene to p53 mutant tumor cells could restore normal regulation of the proliferative and apoptotic signaling pathways. In vitro studies using colon cancer cell lines with known p53 mutations showed that transfection of wild type p53 resulted in inhibition of cell growth and the induction of apoptosis.107,108 Furthermore, replacement of p53 function increases tumor radiation sensitivity and apoptotic response.162,164 In vivo studies using colon cancer xenografts in nude mice showed that the combination of gene therapy and chemotherapy was greater than single agent treatment or controls.164 These data indicate that genetic manipulations, such as wild type p53 gene therapy, can be an effective treatment modality or adjuvant therapy for malignant disease and chemoresistant cancers.
Currently, gene therapy protocols are progressing from preclinical to Phase I clinical trials.164 Despite the advances made, there still remains many potential risks and challenges to the successful application of gene therapy for cancer treatment (Table 4.1). Major challenges lay in the specific targeting of these genes to tumor cells and/or the determination of long-term effects of gene transfer to noncancerous cells. Recent developments in gene therapy involve the establishment of a mutant adenovirus that replicates only in p53-deficient cells.165 This virus will provide specific tumor cell targeting for specific gene therapy. The scientific field of gene therapy is continually evolving, and is an exciting novel approach to the treatment of cancer.
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