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 Ca²⁺, 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®). 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/[³⁵S] 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.     

Isolation of Cancer Stem Like Cells from Human Adenosquamous Carcinoma of the Lung Supports a Monoclonal Origin from a Multipotential Tissue Stem Cell

There is increasing evidence that many solid tumors are hierarchically organized with the bulk tumor cells having limited replication potential, but are sustained by a stem-like cell that perpetuates the tumor. These cancer stem cells have been hypothesized to originate from transformation of adult tissue stem cells, or through re-acquisition of stem-like properties by progenitor cells. Adenosquamous carcinoma (ASC) is an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. The origin of these mixtures is unclear as squamous carcinomas are thought to arise from basal cells in the upper respiratory tract while adenocarcinomas are believed to form from stem cells in the bronchial alveolar junction. We have isolated and characterized cancer stem-like populations from ASC through application of selective defined culture medium initially used to grow human lung stem cells. Homogeneous cells selected from ASC tumor specimens were stably expanded in vitro. Primary xenografts and metastatic lesions derived from these cells in NSG mice fully recapitulate both the adenocarcinoma and squamous features of the patient tumor. Interestingly, while the CSLC all co-expressed cytokeratins 5 and 7, most xenograft cells expressed either one, or neither, with <10% remaining double positive. We also demonstrated the potential of the CSLC to differentiate to multi-lineage structures with branching lung morphology expressing bronchial, alveolar and neuroendocrine markers in vitro. Taken together the properties of these ASC-derived CSLC suggests that ASC may arise from a primitive lung stem cell distinct from the bronchial-alveolar or basal stem cells.     

Gibberellin-photoaffinity labelling of two polypeptides in plant plasma membranes

Two polypeptides of M _r 68 kDa and 18 kDa were gibberellin (GA)-photoaffinity labelled in vitro in plasma membrane preparations from oat ( Avena sativa L.) aleurone and from leaves and stems of wild-type and GA-sensitivity mutants of different species. Labelling of these polypeptides could be competed by biologically active, but not by inactive, GAs, indicating the likely biological significance of these interactions. On 2-dimensional gels the radiolabelled polypeptides were each resolved as one intensely labelled low abundance spot with a slightly lower pI form adjacent to it. There was a strong pH dependency for both labelling events, which correlated well with pH values at which GA are known to be most biologically active. A semi-dwarf GA-sensitivity mutant of sweet pea ( Lathyrus odoratus L.), lb , showed reduced photoaffinity labelling of both polypeptides compared with the wild type, Lb . In the GA-insensitive Arabidopsis thaliana mutant, gai , the level of labelling was the same as in wild type, GAI . This is the first report of GA-binding proteins in plant plasma membranes. Some preliminary sequence data are given for one of the labelled polypeptides. We discuss these mutants and consider their possible roles in GA perception or action.     

The failure of a prostaglandin F2 alpha analogue, cloprostenol, to initiate lactation in cattle

An experiment was designed to determine if the analogue of prostaglandin F2 alpha, cloprostenol, at a dose sufficient to cause luteolysis, was lactogenic in cattle. The mammary glands of eight Friesian heifers were developed by treatment with progesterone plus oestrogen. Lactation was then initiated by administration of cloprostenol and subsequent milk production was compared to that of heifers lactating after a normal pregnancy. Injection of cloprostenol failed to initiate lactation. The level of prolactin but not cortisol in blood was substantially elevated following treatment. The results cast further doubt on the importance of prolactin in the lactogenic process but indicate the likely involvement of glucocorticoids.     

The responsiveness of Avena fatua aleurone protoplasts to gibberellic acid

The sensitivity to gibberellic acid (GA₃) of aleurone protoplasts isolated from a single harvest of an inbred line of Avena fatua seed that had been after-ripened over anhydrous CaCl₂ at 25±2°C and 4±2°C for three years was assessed. Protoplasts isolated from aleurones of seed stored at 25°C produced substantially more -amylase in response to 10^–7 M GA₃ than those isolated from aleurones of seed stored at 4°C. The apparent difference in responsiveness does not appear to be due to a change in the duration of the lag phase between addition of GA₃ and the production of -amylase. The dose response of aleurone protoplasts to GA₃, measured as -amylase production, is complex and appears to have three phases. Protoplasts from seed stored at both temperatures respond appreciably to 10^–14 M GA₃. With increasing concentrations of GA₃, up to 10^–9 M, -amylase production increases similarly in protoplasts from both lots of seed, reaching a level approximately 2.7–3.8 times greater than when no GA₃ is applied. GA₃-induced -amylase production increases markedly as the concentration is raised from 10^–9 M to 10^–6 M, and the response then appears to be saturated. Over this part of the response curve protoplasts from the two seed lots differ markedly in their responsiveness to GA₃. Those from seed stored at 25°C produce considerably more -amylase, >130-fold higher than the minus GA₃ control, than those from seed stored at 4°C, <35-fold higher than the minus GA₃ control. This apparent difference in the responsiveness of aleurone protoplasts to GA₃ could be correlated with the loss of embryo dormancy in seed stored at 25°C. Seed stored at 4°C retained the dormancy characteristics present immediately after harvesting.     

Establishment of human ovarian serous carcinomas cell lines in serum free media

Ovarian cancers are the fifth leading cause of cancer death among US woman. The majority of ovarian cancers belong to a category of serous adenocarcinomas. This type of cancer is often diagnosed at a late stage of the disease. Surgical debulking, followed by chemotherapy is the current treatment. Half of all patients will die within 5 years of diagnosis of the disease. Poor survival may be due to disease progression as a consequence of development of drug resistance, cancer cell heterogeneity within the tumor, or the persistence of cancer stem cells. Cancer stem cells (CSC) are defined as a minority cell type in the tumor, which retains the capacity, through asymmetric division, for self-renewal as well as differentiation into multiple cell types. Through this process, CSC can regenerate the entire tumor phenotype and subsequent metastases. Initial in vitro work in the area of solid tumor CSC biology has focused on the isolation and propagation of cells with CSC-like properties from breast and colon tumors. Breast and colon cell lines with CSC-like properties have been isolated and maintained in vitro for extended periods of time. The in vitro maintenance of these CSC requires growth in hormone-supplemented serum-free media and the use of matrix or growth as tumor spheres (Roberts, Ricci-Vitiani et al., Cammareri et al.). Based on the pioneering work generating breast and colon CSC, our lab has begun to develop methods for the establishment cell lines with CSC-like properties from additional solid tumors. In this article, we describe methods, using defined medium, which allow for the successful establishment of continuous cell cultures from a minority cell type within serous ovarian cancers. The cell lines established using these methods grow in serum-free hormone-supplemented medium either as a monolayer on a matrix, or as tumor spheres in suspension. These cells express markers previously reported for tumor stem cells, including CD44 and CD133, and form tumors that recreate the morphology of the original patient tumor when implanted in immune deficient mice. The introduction of this method will facilitate the expansion of ovarian cancer cells for investigating cancer stem cell biology as well as providing tools to aid in the development of new treatments for this deadly disease.     

Eukaryote polyphosphate kinases: is the 'Kornberg' complex ubiquitous?

Polyphosphate (poly P) is a polymer of up to several hundred phosphate residues and is important to a variety of cell processes. The main poly P synthetic enzyme in many bacteria is poly P kinase 1 (PPK1), which until recently had been detected among eukaryotes in some protists only. There is now evidence for the presence in several other eukaryotes of PPK1 homologues and also a second bacteria-type enzyme, PPK2. The latest genome databases reveal that the 'Kornberg' enzyme complex of three actin-related proteins, termed DdPPK2 in Dictyostelium discoideum, might also be ubiquitous in eukaryotes. Owing to the intimate association of poly P synthesis with the formation of structural fibres, this ubiquity indicates a central role for this molecule in the evolution of eukaryotic cells.