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Biomedical subjects

K M Anderson

Publications and source records attributed to K M Anderson.

226 records · Page 13Linked to original sources
PubMedDate not supplied

Reactive oxygen species and redox-induced programmed cell death due to MK 886: cells ("soil") "trump" agent ("seed").

Micromolar concentrations of the five-lipoxygenase inhibitor, MK 886 induce a "type 1" (apoptotic, extrinsic, death domain, receptor-dependent, caspase-positive) form of programmed cell death in Bcl-2-positive U937 human monoblastoid and HL-60 myeloid leukemia cells. A "type 2" (intrinsic, mitochondria-dependent, autophagic, in some examples caspase-negative (Panc-1)) form is induced in Panc-1 pancreatic and PC3 prostate cell lines. The latter two lines from epithelial-derived solid human cancers are Bcl-2-negative. Micromolar MK 886 induces an acute rise in Ca2+ in washed, Ca2+-poor U937 and HL60 cells in Ca2+ and Mg2+-free Hank's buffer. In U937 cells, much of the increase, or more properly redistribution, is nuclear in location (HL-60 not tested). No MK-886-induced acute Ca2+ increase developed in Panc-1 or PC3 cells. Bcl-2-positive HeLa cervical cancer cells exhibited an acute MK 886-induced increase in Ca2+. In the U937, PC3 and Panc-1 cells examined, MK-886 rapidly increased oxidative stress and decreased mitochondrial membrane potential, indicating that neither event is directly determinative for the altered distribution of Ca2+ or the form of PCD observed. Inhibition of increased U937 Ca2+ by the anti-oxidant, N-acetyl-L-cysteine, the effects of inhibitors of mitochondrial function including antimycin A, atractyloside, cyclosporin A, the L/N channel blocker loperamide, the intracellular chelator BAPTA and 2 agents, HA-14 and 3-methyl-antimycin A3 that impair Bcl-2 function further define these events. These differences in the Ca2+ response and possibly also the form of PCD that results may depend upon the presence of Bcl-2 or a related protein participating in a juxta-nuclear / nuclear Ca2+ ion channel. The role of mitochondria, the mechanism by which increased oxidative stress initiates the rapid release of Ca2+ from intracellular, possibly juxta-nuclear / nuclear sites or its redistribution to U937 Ca2+ nuclei, and whether this "signal" or possibly even ROS themselves mandate the type of PCD observed, presumably by differential modulation of transcription, remain to be determined. Lastly, these results demonstrate that, as might be expected, "soil" (cell type) trumps "seed" (inciting agent)".

Apoptosis↗
PubMedDate not supplied

The proliferative response of hela cells to 2-deoxy-D-glucose under hypoxic or anoxic conditions: an analogue for studying some properties of in vivo solid cancers.

BACKGROUND: Hypoxic cancer cells located beyond the diffusion path of sufficient oxygen are considered a nidus of therapeutic failure. Due to the dependence of many malignantly transformed cells on glycolysis for metabolic energy, inhibiting this and other sources of energy should seriously reduce cell viability and proliferation, additively or even synergistically. MATERIALS AND METHODS: To try and duplicate in vitro some of the features of in vivo cancer cells likely to resist therapy, HeLa cells were incubated with sub-lethal concentrations of 2-deoxy-D-glucose under aerobic, hypoxic or virtually anoxic conditions, verified by increased synthesis of Hif-1alpha, and their replication and survival determined. MK 886, an inhibitor of mitochondrial function was used to estimate participation of that organelle in energy metabolism. RESULTS: Culture of cervical cancer-derived HeLa cells with 2-deoxy-D-glucose under these restrictive conditions did not reduce their proliferation or viability to the expected extent. Their surprisingly robust survival included the anticipated increase in dependence upon glycolysis and implied a likely entrainment of other constitutive and possibly facultative energy sources and pathways. Increased synthesis of Hif-1alpha, increased binding to its consensus sequence and reduced inhibition from MK 886 in cells under oxygen-deficient environments confirmed the presence of restrictive conditions. CONCLUSION: Efforts to suppress HeLa cell survival by reducing glucose consumption and metabolic energy from ambient oxygen may require inhibition of multiple energy sources, possibly not all of them identified. In vitro assessment of agents directed against sources of metabolic energy or of other therapeutic agents against these or additional potential targets should include studies under hypoxia and relative anoxia. In this way, the possible responses of in vivo hypoxic or anoxic cancer cells believed to contribute to therapeutic failure may be estimated.

Cell Hypoxia↗
PubMedDate not supplied

5,8,11,14-eicosatetraynoic acid-induced differentiation of U937 cells.

U937 cells, transformed human monocyte/macrophage precursors, incubated with 5,8,11,14 eicosatetraynoic acid (ETYA), a competitive in vitro and in vivo inhibitor of arachidonic acid, develop properties consistent with a form of cellular differentiation. In order to define the extent of this differentiation and decide whether a monocyte or macrophage-like cell developed, control cells and cells cultured for three days with 40 microns ETYA were examined by transmission electron microscopy. Control cells were characterized by oval nuclei, a high nuclear to cytoplasmic ratio, a cytoplasm with a relative paucity of mitochondria, vesicles and vacuoles, and a Golgi apparatus that was not extensively developed. Ribosomes and putative ribosome-like granules were frequently "grouped" in the cytoplasm, rather than as single granules or in association with the endoplasmic reticulum. The plasma membrane included a discrete region of fimbriated structures that were distinct from pseudopodia. Such structures frequently are expressed in embryonic and transformed cells and may be associated with motility, metastases, with immunologic reactions or with endocytosis. After culture with 40 microns ETYA for three or more days, a morphology consistent with that of immature monocytes developed. These characteristics included lobulated nuclei, a reversal of the nuclear to cytoplasmic ratio, an increased complexity and development of the cytoplasmic components and the disappearance of the fimbriated plasma membrane structures. In addition, "grouped" ribosomes were less evident.(ABSTRACT TRUNCATED AT 250 WORDS)

5,8,11,14-Eicosatetraynoic Acid↗
PubMedDate not supplied

Identification of normal rat organs by two-dimensional protein electrophoresis.

Proteins from nine normal rat organs, including heart, lung thymus, liver, spleen, kidney, prostate, abdominal muscle, and brain, were solubilized, separated by electrophoresis according to their different isoelectric points and molecular weights, and stained with Coomassie blue. Patterns of major proteins unique to each organ were identified. Nine "unknown" samples, chosen from the same sources and submitted for analysis in a single blind study, were easily identified by comparing their protein profiles against the nine "reference" patterns. The ability to identify the origin of a tissue sample without recourse to microscopy, by comparing the pattern of its electrophoresed proteins with a "catalogue" of identified protein profiles, provides a prototype for the identification of histologically indeterminate normal and abnormal cells, tissues, and organs. Application of this technique to problems in human pathology and forensic medicine could prove to be very useful.

Abdominal Muscles↗
PubMedDate not supplied

Progesterone receptor level as a predictor of response to megestrol acetate in advanced breast cancer: a retrospective study.

Megestrol acetate (160 mg/day) produced a response rate of 44% in a retrospective series of 39 evaluable patients with advanced breast cancer. The estrogen-receptor (ER) level was greater than 10 fmols/mg of protein in 28 patients, and the progesterone-receptor (PR) level was greater than 10 fmols/mg of protein in 26 patients. ER and PR levels, age, and disease-free interval were analyzed for their relationship to response. The PR was the single best predictor of response to megestrol acetate; the addition of ER added 2% to the predictive accuracy rate of PR alone.

Breast Neoplasms↗
PubMedDate not supplied

The selective 5-lipoxygenase inhibitor A63162 reduces PC3 proliferation and initiates morphologic changes consistent with secretion.

We examined the effect of A63162 (Abbott), a selective inhibitor of 5-lipoxygenase on human prostate (PC3) cell proliferation. Within 5 min DNA synthesis was reversibly inhibited by 40 microM A63162, without altered cellular attachment or uptake of trypan blue. After 72 Hr, cells continued to be attached and exclude dye, were reduced in number and their histology was altered. Many treated cells were larger, more pleomorphic, with nuclear and cytoplasmic ultrastructural changes consistent with preparation for secretion. Some cells contained moderately swollen, distorted mitochondria. ETYA, a less selective inhibitor of 5-lipoxygenase that also inhibits cell replication, acutely reduced O2 uptake by 40%, but A63162 did not. The retention of the supravital mitochondrial dye, rhodamine 123 was increased by ETYA at 4 hr, but not after 24 hr; retention was not altered by A63162. Although the mechanism by which A63162 reversibly inhibits PC3 proliferation and initiates preparation for secretion is not identified, additional studies should further define its role in these events.

5,8,11,14-Eicosatetraynoic Acid↗
PubMedDate not supplied

An in vivo inhibitor of 5-lipoxygenase, MK886, at micromolar concentration induces apoptosis in U937 and CML cells.

MK886 (Merck Frosst) is a selective in vivo inhibitor of 5-lipoxygenase, active at nanomolar concentrations. At micromolar concentrations, it inhibited the proliferation of U937 monoblastoid cells and of cultured malignant cells from patients with chronic myelogenous leukemia. These cells became morphologically apoptotic, a form of physiologic cell death. U937 cell apoptosis was assessed by flow cytometry, ultrastructure, DNA laddering and immuno-histology for free 3'OH-DNA. MK886-induced apoptosis developed over time as cells were recruited in concert with reduction in their numbers. Some CML cells exhibited cytoplasmic changes of apoptosis without typical nuclear changes. Under conditions used for measuring Ca2+ with Fura 2, 10 micromolar MK886 increased U937 intracellular Ca2+ 4-fold or more over the 8 minute period of measurement. Since MK886 inhibits the association of arachidonic acid with the 5-lipoxygenase activating protein, altered arachidonic acid metabolism may have contributed to these results.

Apoptosis↗
PubMedDate not supplied

Five-lipoxygenase inhibitors reduce Panc-1 survival: the mode of cell death and synergism of MK886 with gamma linolenic acid.

The 5-lipoxygenase inhibitors ETYA, SC41661A and MK886 reduced the proliferation and viability of Panc-1 human pancreatic cancer cells. The extent of inhibition depended upon drug concentration, and with continued culture, cells detached and stained with trypan blue. Although results from flow cytometry were those associated with programmed cell death, despite repeated attempts, no DNA laddering consistent with its later stages was detected, and studies with the TUNEL assay were negative. Light and electron microscopy of cells cultured with SC41661A provided morphologic evidence of a population of "dark" cells and of an incompletely expressed type 1 programmed cell death including margination of chromatin at the nuclear membrane and by consolidation and degeneration of cytoplasmic organelles, along with extensive vacuolization. Cells cultured with MK886 exhibited compact "dark" cells and an unusual cytoplasmic mode of cell death characterized by vacuolization and widely separated smooth internal membranes without diagnostic nuclear changes. This is in marked contrast to the extensive type 1 PCD induced by 5-lipoxygenase inhibitors cultured with human U937 monoblastoid cells. On balance, the response of Panc-1 cells to MK886 suggests expression of a variant type 2 (autophagic) cellular suicide, although some contribution from components of a "cytoplasmic" (type 3?) form of non-necrotic cell death may also be considered. In a European clinical trial, gamma linolenic acid, a polyunsaturated fatty acid that generates free radicals has been combined with 5-fluorouracil as chemotherapy for pancreatic cancer. Panc-1 cell proliferation was insensitive to inhibition by several chemotherapeutic agents employed clinically, including 5-fluorouracil, cisplatin or gemcitabine and only somewhat sensitive to GLA. When gamma linolenic acid was combined with MK886, the more effective of the two 5-lipoxygenase inhibitors, a synergistic reduction in Panc-1 cell number and viability occurred.

Apoptosis↗
PubMedDate not supplied

NTBN, a free radical spin trap induces programmed cell death in human pancreatic cancer (PANC-1) cells.

N-tertiary butyl-a-phenylnitrone, a free radical spin trap at > or = 10 mM concentration, inhibited proliferation and reduced the viability of human pancreatic cancer (Panc-1) cells. The drug concentration determined the extent of inhibition, and with continued culture a proportion of the cells detached, most of which stained with trypan blue. Although hypodiploid cells were detected by flow cytometry of cells cultured with 20 mM NTBN, DNA laddering was absent and the TUNEL reaction negative. "Dark" cells present in samples cultured with 10 mM NTBN exhibited decreased cytoplasmic volume and increased staining with methylene blue and azure II, but lacked characteristic nuclear changes of type 1 programmed cell death. Cells cultured with > 10 mM of the spin trap exhibited nuclear and cytoplasmic changes more consistent with a non-type 1, type 2 variant of PCD with extensive cytoplasmic vacuolization. Careful analysis revealed evidence of marked pinocytosis in some cells. In view of the spin-trap associated pinocytosis, augmented uptake of chemotherapy in affected cells might be anticipated, but additive, synergistic or antagonistic interactions between NTBN and 5-fluorouracil were not observed.

Antimetabolites, Antineoplastic↗