RNA disruption is a widespread phenomenon associated with stress-induced cell death in tumour cells.

We have previously shown that neoadjuvant chemotherapy can induce the degradation of tumour ribosomal RNA (rRNA) in patients with advanced breast cancer, a phenomenon we termed "RNA disruption". Extensive tumour RNA disruption during chemotherapy was associated with a post-treatment pathological complete response and improved disease-free survival. The RNA disruption assay (RDA), which quantifies this phenomenon, is now being evaluated for its clinical utility in a large multinational clinical trial. However, it remains unclear if RNA disruption (i) is manifested across many tumour and non-tumour cell types, (ii) can occur in response to cell stress, and (iii) is associated with tumour cell death. In this study, we show that RNA disruption is induced by several mechanistically distinct chemotherapy agents and report that this phenomenon is observed in response to oxidative stress, endoplasmic reticulum (ER) stress, protein translation inhibition and nutrient/growth factor limitation. We further show that RNA disruption is dose- and time-dependent, and occurs in both tumourigenic and non-tumourigenic cell types. Northern blotting experiments suggest that the rRNA fragments generated during RNA disruption stem (at least in part) from the 28S rRNA. Moreover, we demonstrate that RNA disruption is reproducibly associated with three robust biomarkers of cell death: strongly reduced cell numbers, lost cell replicative capacity, and the generation of cells with a subG1 DNA content. Thus, our findings indicate that RNA disruption is a widespread phenomenon exhibited in mammalian cells under stress, and that high RNA disruption is associated with the onset of cell death.

Chemotherapy and Inflammatory Cytokine Signalling in Cancer Cells and the Tumour Microenvironment.

Cancer is the result of a cell's acquisition of a variety of biological capabilities or 'hallmarks' as outlined by Hanahan and Weinberg. These include sustained proliferative signalling, the ability to evade growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and the ability to invade other tissue and metastasize. More recently, the ability to escape immune destruction has been recognized as another important hallmark of tumours. It is suggested that genome instability and inflammation accelerates the acquisition of a variety of the above hallmarks. Inflammation, is a product of the body's response to tissue damage or pathogen invasion. It is required for tissue repair and host defense, but prolonged inflammation can often be the cause for disease. In a cancer patient, it is often unclear whether inflammation plays a protective or deleterious role in disease progression. Chemotherapy drugs can suppress tumour growth but also induce pathways in tumour cells that have been shown experimentally to support tumour progression or, in other cases, encourage an anti-tumour immune response. Thus, with the goal of better understanding the context under which each of these possible outcomes occurs, recent progress exploring chemotherapy-induced inflammatory cytokine production and the effects of cytokines on drug efficacy in the tumour microenvironment will be reviewed. The implications of chemotherapy on host and tumour cytokine pathways and their effect on the treatment of cancer patients will also be discussed.

Inflammatory cytokine production in tumor cells upon chemotherapy drug exposure or upon selection for drug resistance.

Tumor Necrosis Factor alpha (TNF-α) has been shown to be released by tumor cells in response to docetaxel, and lipopolysaccharides (LPS), the latter through activation of toll-like receptor 4 (TLR4). However, it is unclear whether the former involves TLR4 receptor activation through direct binding of the drug to TLR4 at the cell surface. The current study was intended to better understand drug-induced TNF-α production in tumor cells, whether from short-term drug exposure or in cells selected for drug resistance. ELISAs were employed to measure cytokine release from breast and ovarian tumor cells in response to several structurally distinct chemotherapy agents and/or TLR4 agonists or antagonists. Drug uptake and drug sensitivity studies were also performed. We observed that several drugs induced TNF-αrelease from multiple tumor cell lines. Docetaxel-induced cytokine production was distinct from that of LPS in both MyD88-positive (MCF-7) and MyD88-deficient (A2780) cells. The acquisition of docetaxel resistance was accompanied by increased constitutive production of TNF-αand CXCL1, which waned at higher levels of resistance. In docetaxel-resistant MCF-7 and A2780 cell lines, the production of TNF-α could not be significantly augmented by docetaxel without the inhibition of P-gp, a transporter protein that promotes drug efflux from tumor cells. Pretreatment of tumor cells with LPS sensitized MyD88-positive cells (but not MyD88-deficient) to docetaxel cytotoxicity in both drug-naive and drug-resistant cells. Our findings suggest that taxane-induced inflammatory cytokine production from tumor cells depends on the duration of exposure, requires cellular drug-accumulation, and is distinct from the LPS response seen in breast tumor cells. Also, stimulation of the LPS-induced pathway may be an attractive target for treatment of drug-resistant disease.

SU1498, an inhibitor of vascular endothelial growth factor receptor 2, causes accumulation of phosphorylated ERK kinases and inhibits their activity in vivo and in vitro.

SU1498, an inhibitor of vascular endothelial growth factor receptor 2, has been used successfully to study the physiological manifestations of receptor functions. Here we report that in addition to its anti-receptor activity, SU1498 stimulates accumulation of phosphorylated ERKs in human umbilical vein endothelial cells and in human aortic endothelial cells in a manner that is dependent on the functioning of the upstream components of the MAPK pathway, B-Raf, and MEK kinases. The enhanced accumulation of phospho-ERKs is observed only in cells that have been stimulated with sphingosine 1-phosphate or protein growth factors; SU1498 by itself is ineffective. We show that the inhibitor acts by blocking the kinase activity of phospho-ERK both in a direct assay and in immunoprecipitates from cells treated with the compound. The data reveal a novel and unique way in which MAPK signaling pathway may be blocked in human endothelial cells.

Comparative analysis of in vitro angiogenic activities of endothelial cells of heterogeneous origin.

Endothelial cells are dynamic participants in many aspects of host defense, innate immunity, inflammation, angiogenesis, and vasculogenesis, but the interpretation of studies of their responses is often clouded by the source of the cells under observation. Thus, it is not clear which endothelial cell type should be utilized in in vitro studies to clarify the basis and physiological relevance of essential processes, including chemotactic migration and morphogenic differentiation. In this study, we compared responses of endothelial cells of a variety of origins, as well as an immortalized cell line, using both protein growth factors and biologically active lipid mediators as agonists. While cells of divergent origin displayed marked differences in the extent of their responsiveness, with a few notable exceptions, their pattern of responsiveness to receptor-dependent stimuli was remarkably similar. Moreover, even the immortalized endothelial cell line Py-4-l migrated in a pattern consistent to that seen with primary cells in culture although the immortalized cells failed to form capillarylike structures under any of the conditions tested. We conclude that although the immortalized endothelial cell line Py-4-l is not appropriate for investigations of endothelial cell morphogenic responses, cultured cells from other sources, including arteries, veins, and capillaries, often provide qualitatively similar results to divergent metabolic stimuli.

Migration of vascular smooth muscle cells induced by sphingosine 1-phosphate and related lipids: potential role in the angiogenic response.

The bioactive lipids sphingosine 1-phosphate (SPP), sphingosylphosphorylcholine, and lysophosphatidic acid play an important role in angiogenesis as a result of their effects on both the migration of endothelial cells (ECs) and the integrity of EC monolayers. Here we show that extremely low concentrations of serum and nanomolar concentrations of these biologically active lipids stimulate migration of human aortic smooth muscle cells (SMCs). However, at dosages most effective in promoting EC migration and in enhancing EC monolayer integrity, serum and SPP potently inhibited SMC migration; SPP also blocked the migration induced by protein growth factors. Treatment of SMCs with SPP induced transient phosphorylation of a 175- to 185-kDa protein corresponding to the PDGF receptor, indicating transactivation of this receptor. SPP and related lipids may play a key role in angiogenesis by coordinating the migration of both endothelial cells and vascular smooth muscle cells in response to the changing gradients of these bioactive lipid messengers.