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Virtually all anticancer drugs induce apoptosis in susceptible cell types. This biochemically distinct form of cell death reflects, in large part, activation of caspases, a distinct family of intracellular cysteine proteases. At least two separable processes, one starting with ligation of specific cell surface receptors (so-called death receptors) and the other involving release of cytochrome c from mitochondria, result in transduction of various signals into caspase activity. The mitochondrial or intrinsic pathway appears to play a predominant role in the apoptotic response to anticancer drugs. Bcl-2 family members regulate this pathway by modulating the release of key proapoptotic polypeptides, including cytochrome c and second mitochondrial activator of caspases (Smac)/direct inhibitor of apoptosis (IAP)-binding protein with low pI (DIABLO), from mitochondria. Several Bcl-2 family members that facilitate mitochondrial permeabilization are transcriptional targets of the p53 tumor suppressor gene, providing a partial explanation for the ability of DNA-damaging agents to induce apoptosis. Other proapoptotic Bcl-2 family members are released from cytoskeletal sites upon treatment with paclitaxel or loss of adherence. The antiapoptotic protein XIAP (X chromosome-linked IAP) binds procaspase 9 and prevents its activation. The gene encoding XIAP is activated by nuclear factor-κB, contributing to the antiapoptotic effects of this transcription factor. In this chapter, the various components of the intrinsic pathway are reviewed, alterations in this pathway in various cancers are described, and evidence that some of these same antiapoptotic alterations might contribute to anticancer drug resistance under certain circumstances is discussed. [ABSTRACT FROM AUTHOR]

Even though current therapy is capable of inducing remissions in 60-80% of adults with acute leukemia (1,2), the vast majority of patients are not cured (3-5). This is particularly true for the elderly, who account for the majority of cases of acute myelogenous leukemia (AML) (6,7). Because of unrelated medical problems, as well as a higher incidence of certain resistance mechanisms (e.g., overexpression of the mdr1 multidrug transporter), eradicating AML in these patients has been particularly problematic (8,9). These clinical observations highlight the need for new agents to treat AML. [ABSTRACT FROM AUTHOR]

Western blotting (also called immunoblotting) is a widely utilized laboratory procedure that involves formation and detection of antibody-antigen complexes between antibodies that are initially in solution and antigens that are immobilized on derivatized paper (reviewed in refs. 1-3). This procedure is most commonly performed by sequentially subjecting a complex mixture of polypeptides to three manipulations: (1) electrophoretic separation, usually through polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS); (2) electrophoretic transfer of the separated polypeptides to thin sheets of nitrocellulose or polyvinylidene fluoride (PVDF); and (3) reaction of the sheets sequentially with one or more antibody-containing solutions. Because these are laborious, low-throughput procedures, there has been considerable interest over the past 15 yr in increasing the information gained from immunoblots in various ways. The approaches to this problem have included removal of bound antibodies so that immobilized polypeptides can be reprobed with additional antisera (last reviewed in ref. 4), the sequential (5) or simultaneous (6) use of multiple antisera to detect different antigens, and the development of higher density filter manifolds to permit more spots to be placed in the same area when purified proteins are applied to filters by spot adsorption. After a brief review of critical parameters for successful immunoblotting, some of these approaches are briefly discussed and illustrated. [ABSTRACT FROM AUTHOR]

The quantitation of protein in biological samples is of great importance and utility in many research laboratories. Protein measurements are widely utilized to ensure equal loading of samples on sodium dodecyl sulfide (SDS)-polyacrylamide gels and to provide a basis for comparison of enzyme activities and other analytes. [ABSTRACT FROM AUTHOR]

Western blotting (reviewed in refs. 1-3) refers to formation and detection of an antibody—antigen complex between an antibody and a polypeptide that is immobilized on denvatized paper. Most commonly, polypeptides in a complex mixture are separated by electrophoresis through polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS), electrophoretically transferred to thin sheets of nitrocellulose or nylon, and reacted sequentially with one or more antibody-containing solutions. This sequence of manipulations can be utilized to determine whether a polypeptide recognized by a specific antiserum is present in a particular biological sample (cell type, subcellular fraction, or biological fluid), to follow the purification of the polypeptide, or to assess the location of epitopes within the polypeptide after chemical or enzymatic degradation. If suitable antibodies are available, this same approach can be utilized to search for proteins that bear a particular physiological or pathological posttranslational modification (e.g., refs. 4-6). Alternatively, the same procedure can be utilized to determine whether antibodies that recognize a particular polypeptide are detectable in a sample of biological fluid. Because Western blotting takes advantage of the power of electrophoresis for separating complex mixtures of polypeptides, it is possible to derive large amounts of information from this technique without necessarily purifying the antigen being studied. [ABSTRACT FROM AUTHOR]

Western blotting has been widely utilized to detect various polypeptides or polypeptide epitopes (e.g., posttranslational modifications) within cells (reviewed in 1-4). If the signals in samples being analyzed are compared to a suitable standard curve and appropriate internal standards are utilized to confirm equivalent loading of various samples, Western blotting appears to be a suitable method of quantitating polypeptides as well. [ABSTRACT FROM AUTHOR]