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A large proportion of anti-cancer drugs act by causing chemical modifications to DNA, often involving the addition of part of the drug molecule to DNA to form DNA adducts. As discussed in Chapter 1, resistance mechanisms can act to diminish the extent of drug-target access or to alter the responses of cells to a given level of target modification. Measurement of the extent of drug-DNA interaction permits direct analysis of the contribution of the former type of mechanisms. Measurement of drug-DNA adduct levels can also be important for experiments focused on cellular responses because, first, it may be necessary to prove that observed differences in response are not owing to variation in drug-DNA access. Second, such measurements can be used to measure directly the rate of DNA repair processes, and hence, the influence on these processes of other variables, such as expression of specific genes. Finally, a knowledge of the DNA adducts levels formed in patients during therapy permits direct assessment of the clinical relevance of the levels of DNA modification employed in in vitro experiments. This could be important because the nature and significance of the various types of cellular responses will be dependent on the level of drug-target interaction initiating those responses. [ABSTRACT FROM AUTHOR]

Drug resistance is a major factor that limits the effectiveness of cancer chemotherapy, and there is considerable evidence to suggest a genetic basis for many drug-resistant phenotypes. A major drawback to many of the conventional approaches used to investigate drug-resistance mechanisms is that some prior information or guesswork on the changes that have occurred is required, thus necessitating separate reagents to screen each possible change. When analyzing genetic changes for example, screening is limited to the use of gene or region specific probes as in Southern blot or microsatellite analysis. Recently, the molecular cytogenetic techniques of reverse in situ hybridization (REVISH), and its more advanced relative, comparative genomic hybridization (CGH), have been developed for the rapid global detection and mapping of genetic imbalances in tumor genomes (1-7). In REVISH, genomic DNA from the tumor is used as a complex probe and hybridized to normal metaphase chromosomes (6). Genomic sequences amplified in the tumor are then detected as an increased intensity of signal at the normal chromosomal position from which the amplified sequences are derived (1,6,7). For more accurate analysis of both loss and gain of genetic material, CGH is required; however, CGH involves complex fluorescence-comparison techniques and expert knowledge of chromosome identification (2). However, both REVISH and CGH are ideal methods to detect genetic changes associated with the acquisition of drug resistance in tumors (6,8). [ABSTRACT FROM AUTHOR]

Tumor progression is driven by the accumulation of genetic changes, which, in aggregate, confer the malignant phenotype (1). Thus, tumor development proceeds via clonal divergence with selection for cells with, for example, a proliferative advantage, metastatic potential, or drug resistance phenotype (2-5). Although Southern blotting and polymorphic microsatellite markers are invaluable in providing information about the genetic alterations that underlie the development of solid tumors, the spatial relationships between tumor cells are destroyed during tissue processing (6-8). This leads to a loss of information on genetic heterogeneity and small subpopulations and presents an averaging of the genetic changes. Conventional karyotyping would determine both numerical and structural chromosome anomalies, but is largely impractical for the study of solid tumors, owing to the necessity for cell culture to produce metaphase spreads. Fluorescence in situ hybridization (FISH) is a powerful method for the analysis of genetic change in solid tumors (7,9-12). In particular, it allows the visualization of the genetic makeup of individual cells within their histological context (7). In general, FISH uses chromosome and region specific probes to assess rapidly copy number and rearrangements of chromosomes and genes. The genetically abnormal cells are detected by their aberrant hybridization pattern in the interphase nuclei. Thus, this method of analysis is generally termed "interphase cytogenetics" (9,10,12). [ABSTRACT FROM AUTHOR]

In recent years, immunohistochemistry as applied to the Bcl-2 family of proteins has represented a burgeoning area of interest to cancer researchers. The majority of studies have focused on the original member Bcl-2, first identified via its involvement in the common t(14;18) chromosomal translocation in B-cell lymphomas (1). However, since this discovery, preclinical and clinical interest in Bcl-2 has dramatically increased owing to (a) its recognition as the first of a new class of oncogene able to prolong survival by inhibiting programmed cell death (apoptosis) and (b) the discovery of many additional related genes/proteins some of which, like Bcl-2, inhibit apoptosis, whereas others, such as Bax, conversely promote cell death (2) (Table 1). [ABSTRACT FROM AUTHOR]

The tumor suppressor gene TP53, encoding the p53 protein, has its gene locus on the short arm of chromosome 17 p13.1 (1,2). p53 has been denoted "guardian of the genome" (3) owing to its essential cellular functions in apoptosis control, cell-cycle control, chromosomal segregation, gene transcription, and genomic stability (4). The gene encodes a protein of 393 amino acids (5). The tertiary structure of the p53 protein is known to a relatively large extent; the DNA binding region has been localized to amino acids 102 to 292. Murine double minute-2 (MDM2) binds to the amino terminal of the p53 protein and is a negative regulator of p53 (6). p53 is normally activated by ultraviolet (UV)-light, radiation, cytostatics, and carcinogens. The activation by these may involve interaction with the ataxia telangiectasia gene (ATM). The p53 gene can be inactivated by somatic or germ-line mutations. Somatic mutations in the p53 gene is the most common genetic abnormality so far described in human cancer (7). Patients with germ-line p53 mutation's are part of the Li-Fraumeni syndrome. These patients have an increased risk of developing adrenocortical, breast, gastrointestinal tract, and lung carcinoma, as well as soft-tissue sarcoma and malignant melanoma (8,9). Studies on mice have revealed that induced deficiency of both alleles of the p53 gene is associated within an increased risk of lymphomas and sarcomas (10). p53 can also be inactivated by certain viral oncoproteins, such as human papilloma virus protein E6, SV40 large T-antigen, hepatitis B viral X protein, and adenovirus protein E1B (4). [ABSTRACT FROM AUTHOR]

Alkylating agents exert a wide range of biological effects in both pro- and eukaryotes and there is ever increasing evidence that these effects are mediated via alkylation at the O6position of guanine in DNA (1-4). Repair of such adducts can be mediated by O6-alkylguanine-DNA alkyltransferase (ATase) (3,4). Both pro- and eukaryote ATases transfer alkyl groups from the O6-position of guanine in alkylated DNA (or from other low molecular weight substrates); (5) to a cysteine residue located at the active site of the protein: the reaction is stoichiometric and the protein is autoinactivated (6). This mechanism has been exploited in the design of several different radioactivity-based assays for the enzyme. These involve either measurement of methyl group transfer to protein or the analysis (e.g., by HPLC) of methylated substrate DNA before and after exposure to cell or tissue extracts or restriction endonuclease (RE) site deprotection of synthetic oligonucleotide substrates containing O6-methylguanine. [ABSTRACT FROM AUTHOR]

Microsatellites are simple, tandemly repeated DNA sequences which are abundantly distributed throughout the human genome, and because of their polymorphic nature have been widely utilized as genetic markers (1). They consist of a repeating unit of 1-5 base pairs, averaging 25-60 bases in length, and are commonly found in the form d(CA)n:d(GT)n (2). It has been estimated that there are approximately 100,000 CA/GT repeat sequences in the human genome (3). Studies in colorectal tumors first reported the appearance of instability at microsatellites sequences involving either an expansion or contraction of the repeat sequence (4-6). Such microsatellite instability has now been reported in a variety of different tumor types including lung, breast, stomach, endometrium, and bladder (7-11, reviewed in 12). In addition, a number of other diseases are associated with instability in trinucleotide repeats, such as fragile X syndrome (13), myotonic dystrophy (14) and Huntington's Disease (15). [ABSTRACT FROM AUTHOR]

DNA damaging agents have been widely used in cancer chemotherapy for many years and have proved successful in the treatment of both solid tissue and haematological malignancies. Many commonly used clinical agents, such as members of the nitrogen mustard, chloroethylnitrosourea, dimethane-sulphonate and platinum classes, are bifunctional. DNA interstrand crosslinks (ISC) formed in cells are clearly critical cytotoxic lesions and the formation of DNA ISC has been shown to correlate with cytotoxicity in vitro (1-5). Acquired resistance in vitro to such agents can occur by a number of mechanisms, for example altered drug transport (6), intracellular detoxification via enhanced glutathione and glutathione-S-transferase activity (7), but enhanced DNA repair capacity can also play an important role (3). Clinically the mechanisms of acquired resistance to DNA damaging agents are less clear but enhanced repair of ISC has been suggested to play a role in the acquired resistance of some cancers, e.g., chronic lymphocytic leukaemia to nitrogen mustards (8). In addition, the inherent sensitivity (and curability) of some tumors, e.g., testicular cancer, to DNA damaging agents may result in part from their inability to repair critical DNA lesions (9). [ABSTRACT FROM AUTHOR]

The uracil analog 5-fluorouracil (5-FU) is used as part of combination therapy for the treatment of breast, head/neck, and gastrointestinal malignancies, and has single-agent activity in colorectal cancer. 5-FU itself is inactive and requires intracellular conversion to form cytotoxic nucleotides (1). Several cellular targets for fluoropyrimidines have been well-characterized, including inhibition of thymidylate synthase (TS) by fluorodeoxyuridine monophosphate (FdUMP) and false base incorporation into RNA or DNA. Most investigations into cellular resistance factors regulating 5-FU activity have focused on alterations in (TS) levels and reduced folate pools, the required cofactor for binding dUMP to thymidylate synthase (1). However, the majority of an administered 5-FU dose undergoes metabolism to inactive species through a three-enzyme process, which is initiated and rate-limited by dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2). Following a bolus injection of 5-FU, 80% is degraded via DPD after 24 h after administration (2). Studies of 19F nuclear magnetic resonance (NMR) spectroscopy in mice bearing colon tumors found catabolites made up 51% of labeled drug in the tumor, compared with 26% for the anabolic products (3). [ABSTRACT FROM AUTHOR]

DNA topoisomerases I and II (top1 and top2, respectively) are ubiquitous enzymes that play an essential role in transcription, replication, chromosome segregation, and DNA repair. The basic enzymatic reaction of topoisomerases, namely reversible DNA nicking, is a transesterification reaction where a DNA phosphodiester bond is transferred to a specific enzyme tyrosine residue. Eukaryotic top1 and top2 exhibit major differences concerning their mechanism of action. Top1 acts as a monomer and forms a covalent bond with the 3′-terminus of a DNA single-strand break (1-3) whereas top2 acts as an homodimer and forms a covalent bond with the 5′-terminus of the DNA double-strand break with a four base-pairs overhang (Fig. 1) (1-4). No energy cofactor is required for top1 activity, whereas top2 hydrolyzes adenosine triphosphate (ATP) during its catalytic cycle. [ABSTRACT FROM AUTHOR]

As outlined in Chapter 8, glutathione in the intact cell is maintained predominantly in its reduced form by the cytosolic enzyme, glutathione reductase. Cell lysis can lead to rapid oxidation to the oxidized form, GSSG, and degradation by γ-glutamyl transpeptidase. In order to obtain a true measurement of the amount of reduced glutathione (GSH) in living cells we have utilized the method of Cotgreave and Moldeus (1) in which GSH is derivatized using monobromobimane (MBBr), which can freely cross the cell membrane (Fig. 1). The GSH-MBBr adduct is then extracted and the amount formed measured by high-performance liquid chromatography (HPLC) with fluorescence detection. [ABSTRACT FROM AUTHOR]

Glutathione is an intracellular, nonprotein thiol that appears to play an important role in protection of the cell against cytotoxic drugs (1) and has been implicated in the control of cell cycling (2,3) and apoptosis (4,5). It can exist in an oxidized (disulfide, [GSSG]) and a reduced (sulphydryl, [GSH]) form. In general, GSSG comprises less than 1% of the total intracellular glutathione. In circumstances of oxidative stress, GSH dimerizes to form glutathione disulfide (GSSG), making protons available to neutralize reactive oxygen species (Reaction 1). In vivo, reduction of GSSG is catalysed by glutathione reductase, efficiently regenerating high intracellular GSH levels (Reaction 2) [ABSTRACT FROM AUTHOR]

Accurate determination of the expression of P-glycoprotein and its encoding mRNA, MDR-1, is key to determining the importance of this mechanism of drug resistance in the clinic. This understanding is critical to development of P-glycoprotein antagonists to block drug efflux and to overcome drug resistance. Reversal studies are best performed in diseases in which this mechanism represents the major component of drug resistance. The clearest approach to identifying these is to measure MDR-1 mRNA or P-glycoprotein in tumors at diagnosis, at the time of disease recurrence, and before and after treatment with P-glycoprotein reversal agents. [ABSTRACT FROM AUTHOR]

The MDR1-encoded P-glycoprotein (Pgp), when overexpressed in tumor cells, confers resistance to many clinically important classes of anticancer drugs. This phenomenon is called multidrug resistance (MDR). The finding that this gene was expressed in many types of human cancers has stimulated many studies into the relevance of this protein for clinical chemotherapy resistance (1). Pgp is a protein that causes a net transport of substrate drug molecules over the plasma membrane of the cell, resulting in a lowered free cytosolic drug concentration. Therefore, the drug target(s) "feel" a lower drug concentration, resulting in less drug-induced damage and cell-kill. The measurement of the active (adenosine triphosphate [ATP]-dependent) drug transport or efflux function of Pgp is, therefore, a theoretically elegant way to quantify the number of active or "functional" Pgp molecules per cell (2,3). Such assays are called functional assays in this chapter. One disadvantage is, however, that without additional data no unambiguous evidence of the molecular nature of the transport protein is obtained, the advantage is that the relevant biological feature is measured. The latter cannot be derived easily from the mRNA or protein expression levels, because a number of factors may influence the net effect of transporter proteins, such as other membrane properties specific to certain cells (3,4). In practice, important criteria for the usefulness of any MDR assay are its specificity, sensitivity, and reproducibility. [ABSTRACT FROM AUTHOR]

The recognition over the past decade that apoptosis represents a critical element in cell number control in physiological and pathological situations has been well-reviewed (1-4). In addition there is increasing recognition that many of the effects of chemo- and radiotherapeutic agents are mediated by apoptosis (5-7). The seminal work of Kerr, Wyllie, and Currie (8), building upon the earlier observations of Glucksmann (9) and Saunders (10), should be read by those interested in assaying apoptosis because of the excellent photomicrographs that document the morphological features of the process. This is important, because despite considerable progress in the understanding of the mechanistic basis of apoptosis, morphological analysis remains unquestionably the "gold standard" for its assessment and quantitation. [ABSTRACT FROM AUTHOR]

Flow cytometry allows rapid multiparameter analyses of individual cells. Cells are illuminated with incident laser light of a specific wavelength. Resultant light scatter signals and fluorescence-emission signals from fluorochromes contained at the surface of the cell or within it are detected by an array of photomultiplier tubes. For example, fluorochrome-conjugated antibodies and DNA interchelating dyes can be used to acquire information on protein expression and DNA ploidy (via fluorescence signals) together with information on cell size and structure (via light-scatter signals). [ABSTRACT FROM AUTHOR]

The clonogenic assay described in Chapter 2 is not suitable for all cell lines. Many adherent cell lines do not form colonies and clearly it is not applicable to non-adherent cell lines. Furthermore, it is slow and time consuming. This chapter describes an alternative cytotoxicity assay that has a number of advantages when compared with a clonogenic assay. It is quick and easy and allows a large number of assays to be carried out in one batch. This is an important consideration when making comparisons between cell lines, between cytotoxic agents, or when evaluating combinations of drugs. No one cytotoxicity assay is ideal and it is always advisable to support results with those from alternative assays where possible. [ABSTRACT FROM AUTHOR]

The use of cell culture systems to assess the toxicity of anticancer agents began over 50 years ago following the observation of the antineoplastic effects of nitrogen mustard (1). There are a wide variety of assays designed to evaluate cellular drug sensitivity described in the literature. These assays essentially fall into two groups; those that measure cell survival and those that measure cytotoxicity. Cytotoxicity assays include methods such as trypan blue dye exclusion, 51Cr release and 3H-thymidine incorporation (2-4) and these assays assess the structural integrity and metabolic function of the cells following drug exposure. In contrast, cell survival assays measure the end result of these effects on the cell which can be either cell death or recovery. A cell survival assay thus requires a measure of the ability of cells to proliferate and this is usually an estimate of the ability of individual cells to form colonies. However, cytotoxicity assays can also measure the ability of cells to proliferate if the cells are allowed a period of growth following drug exposure. This recovery time is comparable to the time taken for formation of colonies in a clonogenic assay. [ABSTRACT FROM AUTHOR]

There are very few tumor types in which the use of chemotherapy can bring about prolonged survival, and possibly cure, for individual patients. The most common reason for this is the development of drug resistance within tumor cells. The laboratory study of resistance to anticancer drugs has resulted in the discovery of numerous mechanisms present within tumor cells that act to reduce their cytotoxic effects. However, the failure to translate this basic laboratory research into improved clinical outcome for patients remains one of the most pressing problems in contemporary cancer research. [ABSTRACT FROM AUTHOR]