Safety and antitumor activity of recombinant soluble Apo2 ligand.

TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy. Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis. Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells. To investigate Apo2L's therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein. Several normal cell types were resistant in vitro to apoptosis induction by Apo2L. Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined. Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer. Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence. Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival. Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation. Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.

A novel 96-well scintillation proximity assay for the measurement of apoptosis.

The translocation of phospholipids across the plasma membrane has been widely documented as one of the earliest measurable biochemical events of apoptosis. Using fluorescently labelled annexin V, which preferentially binds phosphatidylserine (PS) in the presence of Ca(2+), the externalization of PS can be measured and apoptosis quantified using flow cytometry. Conventional detection methods utilizing annexin V, while faster than in situ DNA end-labelling or DNA laddering, require extensive sample preparation which may compromise samples and makes rapid, high volume screening prohibitive. This paper describes a novel assay for the measurement of apoptosis based upon binding of radiolabelled annexin V to apoptotic cells attached to the growth surface of a 96-well scintillating microplate (Cytostar-T(R)). We compared measurements of apoptosis made by flow cytometry to those obtained with the scintillating microplate in three model systems, treatment of: mouse connective tissue (L-M) cells with lymphotoxin (LT), human lung carcinoma (H460) cells with Apo-2 ligand and human umbilical vein endothelial (HUVE) cells with staurosporine. In this assay, we compare both direct and indirect labelling methods by utilizing either iodinated annexin V or biotinylated annexin V/[(35)S] streptavidin to radiolabel apoptotic cells. The signal detected is a direct consequence of the binding of annexin V to externalized PS on apoptotic cells and the proximity of the label to the base of the plate. Using this method, separation of bound and unbound radiolabel signal occurs directly within the well resulting in a sensitive assay that requires minimal manipulation and can accomodate a large number of samples.

Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation.

Vascular endothelial growth factor (VEGF) has been found to have various functions on endothelial cells, the most prominent of which is the induction of proliferation and differentiation. In this report we demonstrate that VEGF or a mutant, selectively binding to the Flk-1/KDR receptor, displayed high levels of survival activity, whereas Flt-1-specific ligands failed to promote survival of serum-starved primary human endothelial cells. This activity was blocked by the phosphatidylinositol 3'-kinase (PI3-kinase)-specific inhibitors wortmannin and LY294002. Endothelial cells cultured in the presence of VEGF and the Flk-1/KDR-selective VEGF mutant induced phosphorylation of the serine-threonine kinase Akt in a PI3-kinase-dependent manner. Akt activation was not detected in response to stimulation with placenta growth factor or an Flt-1-selective VEGF mutant. Furthermore, a constitutively active Akt was sufficient to promote survival of serum-starved endothelial cells in transient transfection experiments. In contrast, overexpression of a dominant-negative form of Akt blocked the survival effect of VEGF. These findings identify the Flk-1/KDR receptor and the PI3-kinase/Akt signal transduction pathway as crucial elements in the processes leading to endothelial cell survival induced by VEGF. Inhibition of apoptosis may represent a major aspect of the regulatory activity of VEGF on the vascular endothelium.

Glycerophosphorylethanolamine (GPEA) identified as an hepatocyte growth stimulator in liver extracts.

Extracts from weanling pig liver were found to act synergistically with growth factors such as hepatocyte growth factor and transforming growth factor-alpha to stimulate hepatocyte growth in serum-free cultures. In the absence of added growth factors, the extracts had no activity. The compound responsible for this activity was isolated by passing heat-treated liver extract through anion-exchange and heparin columns followed by gel filtration at neutral and low pH, reversed-phase HPLC, and a final gel filtration column at low pH. The activity was followed throughout the purification by its ability to increase substantially the incorporation of [3H]thymidine into primary rat hepatocytes cultured serum-free in the presence of hepatocyte growth factor. The active compound was identified by NMR and mass spectrometry as glycerophosphorylethanolamine (GPEA), a breakdown product of the phospholipid phosphatidylethanolamine. The ethanolamine portion of the molecule was critical for the observed activity, whereas the glycerol phosphate portion was not necessary. In the absence of added growth factors, neither GPEA nor ethanolamine had any stimulatory effect on the cells. These results demonstrate that hepatocytes grown in culture, and especially those grown in serum-free media, require a supplement of ethanolamine and/or GPEA. In the absence of these compounds, their response to growth stimuli is greatly reduced.

Growth regulation of human breast and ovarian tumor cells by heregulin: Evidence for the requirement of ErbB2 as a critical component in mediating heregulin responsiveness.

Alterations in the expression of the epidermal growth factor (EGF) receptor ErbB family are frequently encountered in a number of human cancers. Two of these receptors, ErbB3 and ErbB4, are known to bind a family of related proteins termed heregulins (HRGs) or neu differentiation factors. In biologically relevant systems, interaction of HRG with ErbB3 or ErbB4 results in the transactivation of ErbB2. In this report, we show that ErbB2 is a critical component in mediating HRG responsiveness in a panel of human breast and ovarian tumor cell lines. Because HRGs have been reported to elicit diverse biological effects on cultured cells, including growth stimulation, growth inhibition, and induction of differentiation, we systematically examined the effect of rHRG beta 1 on tumor cell proliferation. HRG binding studies were performed with a panel of breast and ovarian tumor cell lines expressing a range of levels of ErbB2. The biological responses to HRG were also compared to EGF and to the growth-inhibitory anti-ErbB2 antibody, 4D5. In most cases, HRG stimulation of DNA synthesis correlated with positive effects on cell cycle progression and cell number and with enhancement of colony formation in soft agar. On each cell line tested, the HRG effects were distinguishable from EGF and 4D5. Our findings indicate that HRG induces cell proliferation in a number of tumor cell lines. In addition, we show that methods for measuring cell proliferation, as well as experimental conditions, are critical for determining HRGs effect on tumor cell growth in vitro.