Method validation and preliminary qualification of pharmacodynamic biomarkers employed to evaluate the clinical efficacy of an antisense compound (AEG35156) targeted to the X-linked inhibitor of apoptosis protein XIAP.

Data are presented on pharmacodynamic (PD) method validation and preliminary clinical qualification of three PD biomarker assays. M65 Elisa, which quantitates different forms of circulating cytokeratin 18 (CK18) as putative surrogate markers of both apoptotic and nonapoptotic tumour cell death, was shown to be highly reproducible: calibration curve linearity r2 = 0.996, mean accuracy > 91% and mean precision < 3%, n = 27.

Recent advances in targeting regulators of apoptosis in cancer cells for therapeutic gain.

Apoptosis is a fundamental cellular death process that is essential for normal tissue homeostasis, whose deregulation is associated with several human disease states, including cancer. Increased understanding of cancer biology has led to the hypothesis that although cancer cells are inherently resistant to the engagement of apoptosis due to the deregulation of molecular components of core apoptotic machinery or of survival signalling cascades, they are primed to die as a result of microenvironmental and oncogenic proapoptotic stress. Recently, deeper insight into the molecular regulation of apoptosis and, specifically, into its deregulation in cancer has led to the development of promising therapies to restore apoptosis and enable selective tumour cell kill.

Cell cycle specific induction of HL-60 cell differentiation and apoptosis by mycophenolic acid.

HL-60 cells undergo terminal differentiation and apoptosis in response to different types of sub-toxic and toxic perturbations respectively. The mechanism by which cells sense different amounts of perturbation to activate pathways that lead to the engagement of a relevant biological response is not known. The response of HL-60 cells to treatment with the immunosuppressant mycophenolic acid (MPA), a specific inhibitor of dGTP/GTP-synthesis, allowed quantitation of a metabolic perturbation which triggered a cellular response.

Comparison of phosphatidylinositol-3-kinase signalling within a panel of human colorectal cancer cell lines with mutant or wild-type PIK3CA.

Recent studies have identified conserved missense mutations in PIK3CA, the gene encoding the catalytic phosphatidylinositol-3-kinase subunit p110alpha, in a variety of human cancers. Further investigation demonstrated that PIK3CA mutations lead to increased basal phosphatidylinositol-3-kinase activity, promoting cell growth and invasion [Samuels, Y., Diaz, L.

Expression of pro-apoptotic Bfk isoforms reduces during malignant transformation in the human gastrointestinal tract.

Reduced expression of pro-apoptotic Bcl-2 family proteins has been described in many gastrointestinal cancers, and may play a role in tumourigenesis. The human homologue of the pro-apoptotic Bcl-2 protein, Bfk, is predominantly expressed in tissues of the gastrointestinal tract. In colon, four alternatively spliced isoforms were identified; of which two are pro-apoptotic when overexpressed.

Expression of the proapoptotic protein Bid is an adverse prognostic factor for radiotherapy outcome in carcinoma of the cervix.

The Bcl-2 family of apoptotic regulators is thought to play an essential role in cancer development and influence the sensitivity of tumour cells to radiotherapy. Bid is an abundantly expressed Bcl-2 family protein playing a central role in various pathways of apoptosis by integrating and converging signals at the mitochondria. The relevance of apoptotic modulation by Bcl-2 and related proteins in tumour development and radiation response for human tumours remains undefined.

Validation of pharmacodynamic assays to evaluate the clinical efficacy of an antisense compound (AEG 35156) targeted to the X-linked inhibitor of apoptosis protein XIAP.

The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial.

Construction and characterization of multiple human colon cancer cell lines for inducibly regulated gene expression.

Validation of targets for cancer drug discovery requires robust experimental models. Systems based on inducible gene expression are well suited to this purpose but are difficult to establish in several epithelial cell types. Using the recently discovered transcriptional transactivator (rtTA2S-M2), we developed a strategy for fast and efficient generation of Tet On cells.

Apoptosis pathway-targeted drugs--from the bench to the clinic.

It is an exciting time for cancer researchers in the field of apoptotic cell death. The avalanche of discoveries over the past decade or so regarding how apoptosis is regulated begins to be exploited for therapeutic benefit as the first apoptosis-targeted drugs enter early clinical trials. This chapter provides a selective review on the development of such drugs.

Expression of kinase-defective mutants of c-Src in human metastatic colon cancer cells decreases Bcl-xL and increases oxaliplatin- and Fas-induced apoptosis.

Tumor resistance to current drugs prevents curative treatment of human colon cancer. A pressing need for effective, tumor-specific chemotherapies exists. The non-receptor-tyrosine kinase c-Src is overexpressed in >70% of human colon cancers and represents a tractable drug target.

Role of mitochondrial membrane permeabilization in apoptosis and cancer.

The release of proteins from the intermembrane space of mitochondria is one of the pivotal events in the apoptotic process, which can lead to the activation of caspases and the ultimate demise of the cell. How these proteins exit the mitochondria is still a matter of intense debate. Here, we discuss the possible mechanisms behind the release of apoptogenic proteins, the ways in which cancer cells subvert these mechanisms, and the therapeutic regimens that aim to promote the timely loss of integrity of the outer mitochondrial membrane.

Hypoxia-mediated down-regulation of Bid and Bax in tumors occurs via hypoxia-inducible factor 1-dependent and -independent mechanisms and contributes to drug resistance.

Solid tumors with disorganized, insufficient blood supply contain hypoxic cells that are resistant to radiotherapy and chemotherapy. Drug resistance, an obstacle to curative treatment of solid tumors, can occur via suppression of apoptosis, a process controlled by pro- and antiapoptotic members of the Bcl-2 protein family. Oxygen deprivation of human colon cancer cells in vitro provoked decreased mRNA and protein levels of proapoptotic Bid and Bad.

Proapoptotic Bid binds to monolysocardiolipin, a new molecular connection between mitochondrial membranes and cell death.

Recent evidence indicates that the mitochondrial lipid cardiolipin may be instrumental in the proapoptotic action of Bcl-2 family proteins on mitochondrial membranes, leading to the release of apoptogenic factors. However, contrasting evidence indicates that progressive loss of cardiolipin occurs during apoptosis. Here we show that Bid, a crucial proapoptotic protein that integrates the action of other Bcl-2 family members, exhibits discrete specificity for metabolites of cardiolipin, especially monolysocardiolipin (MCL).

Recent advances in understanding apoptosis: new therapeutic opportunities in cancer chemotherapy.

Major advances have been made in our understanding of the regulation of the molecular machinery of apoptosis in vitro. Molecules linking proliferation and apoptosis in healthy cells are being identified and here apoptotic cell death provides the 'fail-safe' mechanism to counteract excess proliferation. More recently, pioneering work on the regulation of apoptosis, in animal models of tumour development, has shown that suppression of apoptosis in the presence of a proliferative stimulus is sufficient for tumour development.

Mitochondrial membrane permeabilisation by Bax/Bak.

Recent studies on cells derived from mice deficient in both multi-domain pro-apoptotic genes of the Bcl-2 family, Bax and Bak, suggest that one or other of these proteins are required for the release of apoptogens such as cytochrome c from mitochondria. In addition BH-3 only proteins of this family such as Bid are suggested to act as critical death inducing ligands via interactions with pro- and anti-apoptotic Bcl-2 family proteins with Bax or Bak at the mitochondrial surface. Despite this increase in knowledge it remains unclear precisely how Bak and Bax promote outer mitochondrial membrane (OMM) permeabilisation.

Post-translational modification of Bid has differential effects on its susceptibility to cleavage by caspase 8 or caspase 3.

Bid is instrumental in death receptor-mediated apoptosis where it is cleaved by caspase 8 at aspartate 60 and aspartate 75 to generate truncated Bid (tBID) forms that facilitate release of mitochondrial cytochrome c. Bid is also cleaved at these sites by caspase 3 that is activated downstream of cytochrome c release after diverse apoptotic stimuli. In this context, tBid may amplify the apoptotic process.

Cellular damage signals promote sequential changes at the N-terminus and BH-1 domain of the pro-apoptotic protein Bak.

The pro-apoptotic protein Bak is converted from a latent to an active form by damage-induced signals. This process involves an early exposure of an occluded N-terminal epitope of Bak in intact cells. Here we report a subsequent damage-induced change in Bak, detected using an antibody to the central BH-1 domain.

Damage-induced Bax N-terminal change, translocation to mitochondria and formation of Bax dimers/complexes occur regardless of cell fate.

Sequential steps in the activation of the pro-apoptotic protein Bax are described for cells with different sensitivity to cytotoxins. SH-EP1 and SH-SY5Y human neuroblastoma cells, derived from a single precursor cell line, differed in their sensitivity to taxol but showed the same sensitivity to cisplatin. Both drugs, in both cell lines, induced exposure of a constitutively occluded N-terminal epitope of Bax.

Apoptosis and cancer chemotherapy.

The explosion of interest in apoptosis amongst cancer biologists has been underpinned by the hope that a mechanistic understanding of cell death will inform our understanding of tumour drug resistance. A framework for drug-induced apoptosis can now be described in which a balance exists between intrinsic and extrinsic survival signals and drug-induced death signals. Pro- and anti-apoptotic signals impact upon pro-apoptotic members of the Bcl-2 family of proteins, which ultimately control the cellular fate.

Bid, a widely expressed proapoptotic protein of the Bcl-2 family, displays lipid transfer activity.

Bid is an abundant proapoptotic protein of the Bcl-2 family that is crucial for the induction of death receptor-mediated apoptosis in primary tissues such as liver. Bid action has been proposed to involve the relocation of its truncated form, tBid, to mitochondria to facilitate the release of apoptogenic cytochrome c. The mechanism of Bid relocation to mitochondria was unclear.

Modulating sensitivity to drug-induced apoptosis: the future for chemotherapy?

Drug resistance is a fundamental problem in the treatment of most common human cancers. Our understanding of the cellular mechanisms underlying death and survival has allowed the development of rational approaches to overcoming drug resistance. The mitogen activated protein kinase family of protein serine/threonine kinases has been implicated in this complex web of signalling, with some members acting to enhance death and other members to prevent it.

Bcr-Abl protein tyrosine kinase activity induces a loss of p53 protein that mediates a delay in myeloid differentiation.

Chronic myeloid leukaemia is a haemopoietic stem cell disorder, the hallmark of which is the expression of the Bcr-Abl Protein Tyrosine Kinase (PTK). We have previously reported that activation of a temperature sensitive Bcr-Abl PTK in the multipotent haemopoietic cell line FDCP-Mix for short periods resulted in subtle changes including, a transient suppression of apoptosis and no inhibition of differentiation. In contrast, activation of the Bcr-Abl PTK for 12 weeks results in cells that display a delay in differentiation at the early granulocyte stage.

v-Abl protein-tyrosine kinase up-regulates p21WAF-1 in cell cycle arrested and proliferating myeloid cells.

v-Abl protein-tyrosine kinase (PTK) promotes cell survival without cell proliferation in interleukin (IL)-3-deprived IC.DP premast cells (1). We now show that in these conditions v-Abl PTK transcriptionally up-regulated the cyclin-dependent kinase inhibitor (CDKI) p21(WAF-1) and inhibited CDK2 and CDK4.