Comparability Considerations and Challenges for Expedited Development Programs for Biological Products.

Expedited development programs for biological products to be used in the treatment of serious conditions bring about challenges because of the compressed clinical development timeframes. As expedited development does not lessen the quality expectations, one challenge is providing adequate chemistry, manufacturing, and control (CMC) information required to support approval of a biological product. In particular, the analytical comparability and, in some cases, pharmacokinetic comparability studies needed to bridge the clinical material to the commercial material could delay submission of applications for life-saving medicines.

RhoGDI deficiency induces constitutive activation of Rho GTPases and COX-2 pathways in association with breast cancer progression.

Rho GDP Dissociation Inhibitor (RhoGDI) is a key regulator of Rho GTPases. Here we report that loss of RhoGDI significantly accelerated xenograft tumor growth of MDA-MB-231 cells in animal models. At the molecular level, RhoGDI depletion resulted in constitutive activation of Rho GTPases, including RhoA, Cdc42, and Rac1.

Ricin detection: tracking active toxin.

Ricin is a plant toxin with high bioterrorism potential due to its natural abundance and potency in inducing cell death. Early detection of the active toxin is essential for developing appropriate countermeasures. Here we review concepts for designing ricin detection methods, including mechanism of action of the toxin, advantages and disadvantages of current detection assays, and perspectives on the future development of rapid and reliable methods for detecting ricin in environmental samples.

The use of a stably expressed FRET biosensor for determining the potency of cancer drugs.

Many cancer drugs are intended to kill cancer cells by inducing apoptosis. However, the potency assays used for measuring the bioactivity of these products are generally cell viability assays which do not distinguish between cell death and growth inhibition. Here we describe a cell-based fluorescence resonance energy transfer (FRET) biosensor designed to measure the bioactivity of apoptosis inducing cancer drugs.

Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5.

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. We have previously shown that deficiency of DR4 and DR5 on the surface membrane is a critical mechanism of cancer cell resistance to the recombinant human TRAIL and its receptor agonistic antibodies, which are being evaluated clinically for treating cancers. In certain cancer cells, DR4 and DR5 were found to be mislocalized in intracellular compartments yet to be characterized.

Mislocalization of death receptors correlates with cellular resistance to their cognate ligands in human breast cancer cells.

Multiple clinical trials are ongoing to evaluate the potential antitumor activity of human TNF variants, Fas ligand (FasL), TNF-related apoptosis inducing ligand (TRAIL) and its agonistic antibodies. These drug products act through the death receptors (DRs) TNF receptor 1 (TNFR1), Fas/CD95, DR4 (TRAIL-R1) and/or DR5 (TRAIL-R2), respectively. Therefore, characterization of the level and localization of DR expression in cancer cells is important for DR-targeted therapy.

Association between α4 integrin cytoplasmic tail and non-muscle myosin IIA regulates cell migration.

α4β1 integrin regulates cell migration via cytoplasmic interactions. Here, we report an association between the cytoplasmic tail of α4 integrin (α4 tail) and non-muscle myosin IIA (MIIA), demonstrated by co-immunoprecipitation of the MIIA heavy chain (HC) with anti-α4-integrin antibodies and pull-down of MIIA-HC with recombinant α4 tail from cell lysates. The association between the α4 tail and MIIA does not require paxillin binding or phosphorylation at Ser988 in the α4 tail.

Association of D4-GDI expression with breast cancer progression.

D4-GDI is a key regulator of Rho GTPases that have been implicated in several aspects of breast tumorigenesis. We have previously found that D4-GDI was selectively expressed in breast cancer cell lines over normal mammary epithelial cells [45]. In this study, we investigated the expression level of D4-GDI in breast tumor specimens (n = 165) by immunohistochemistry using a validated antibody that specifically recognizes the full-length D4-GDI protein.

Blockade of Rac1 activity induces G1 cell cycle arrest or apoptosis in breast cancer cells through downregulation of cyclin D1, survivin, and X-linked inhibitor of apoptosis protein.

Rac1 GTPase regulates a variety of signaling pathways that are implicated in malignant phenotypes. Here, we show that selective inhibition of Rac1 activity by the pharmacologic inhibitor NSC23766 suppressed cell growth in a panel of human breast cancer cell lines, whereas it had little toxicity to normal mammary epithelial cells. NSC23766 elicits its cytotoxicity via two distinct mechanisms in a cell line-dependent manner: induction of G(1) cell cycle arrest in cell lines (MDA-MB-231, MCF7, and T47D) that express retinoblastoma (Rb) protein or apoptosis in Rb-deficient MDA-MB-468 cells.

Repeated treatment with subtoxic doses of TRAIL induces resistance to apoptosis through its death receptors in MDA-MB-231 breast cancer cells.

Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is being evaluated clinically in treating various malignancies. Previous studies have shown that repeated application of high doses of rhTRAIL results in a subpopulation of parental cells that is unresponsive to the death ligand. However, it is not clear whether TRAIL-sensitive cancer cells could acquire resistance to TRAIL treatment.

Silencing of D4-GDI inhibits growth and invasive behavior in MDA-MB-231 cells by activation of Rac-dependent p38 and JNK signaling.

The Rho GDP dissociation inhibitor D4-GDI is overexpressed in some human breast cancer cell lines (Zhang, Y., and Zhang, B. (2006) Cancer Res.

alpha4 beta1-Integrin regulates directionally persistent cell migration in response to shear flow stimulation.

alpha(4)beta(1)-Integrin plays a pivotal role in cell migration in vivo. This integrin has been shown to regulate the front-back polarity of migrating cells via localized inhibition of alpha(4)-integrin/paxillin binding by phosphorylation at the alpha(4)-integrin cytoplasmic tail. Here, we demonstrate that alpha(4)beta(1)-integrin regulates directionally persistent cell migration via a more complex mechanism in which alpha(4)-integrin phosphorylation and paxillin binding act via both cooperative and independent pathways.