Marian L. Lewis

Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat)

Alteration in cytoskeletal organization appears to underlie mechanisms of gravity sensitivity in space‐flown cells. Human T lymphoblastoid cells (Jurkat) were flown on the Space Shuttle to test the hypothesis that growth responsiveness is associated with microtubule anomalies and mediated by apoptosis. Cell growth was stimulated in microgravity by increasing serum concentration. After 4 and 48 h, cells filtered from medium were fixed with formalin. Post‐flight, confocal microscopy revealed diffuse, shortened microtubules extending from poorly defined microtubule organizing centers (MTOCs). In comparable ground controls, discrete microtubule filaments radiated from organized MTOCs and branched toward the cell membrane. At 4 h, 30% of flown, compared to 17% of ground, cells showed DNA condensation characteristic of apoptosis. Time‐dependent increase of the apoptosis‐associated Fas/APO‐1 protein in static flown, but not the in‐flight 1 g centrifuged or ground controls, confirmed microgravity‐associated apoptosis. By 48 h, ground cultures had increased by 40%. Flown populations did not increase, though some cells were cycling and actively metabolizing glucose. We conclude that cytoskeletal alteration, growth retardation, and metabolic changes in space‐flown lymphocytes are concomitant with increased apoptosis and time‐dependent elevation of Fas/APO‐1 protein. We suggest that reduced growth response in lymphocytes during spaceflight is linked to apoptosis.—Lewis, M. L., Reynolds, J. L., Cubano, L. A., Hatton, J. P., Lawless, B. D., Piepmeier, E. H. Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat). FASEB J. 12, 1007–1018 (1998)

Spaceflight and clinorotation cause cytoskeleton and mitochondria changes and increases in apoptosis in cultured cells.

The cytoskeleton is a complex network of fibers that is sensitive to environmental factors including microgravity and altered gravitational forces. Cellular functions such as transport of cell organelles depend on cytoskeletal integrity; regulation of cytoskeletal activity plays a role in cell maintenance, cell division, and apoptosis. Here we report cytoskeletal and mitochondria alterations in cultured human lymphocyte (Jurkat) cells after exposure to spaceflight and in insect cells of Drosophila melanogaster (Schneider S-1) after exposure to conditions created by clinostat rotation. Jurkat cells were flown on the space shuttle in Biorack cassettes while Schneider S-1 cells were exposed to altered gravity forces as produced by clinostat rotation. The effects of both treatments were similar in the different cell types. Fifty percent of cells displayed effects on the microtubule network in both cell lines. Under these experimental conditions mitochondria clustering and morphological alterations of mitochondrial cristae was observed to various degrees after 4 and 48 hours of culture. Jurkat cells underwent cell divisions during exposure to spaceflight but a large number of apoptotic cells was also observed. Similar results were obtained in Schneider S-1 cells cultured under clinostat rotation. Both cell lines displayed mitochondria abnormalities and mitochondria clustering toward one side of the cells which is interpreted to be the result of microtubule disruption and failure of mitochondria transport along microtubules. The number of mitochondria was increased in cells exposed to altered gravity while cristae morphology was severely affected indicating altered mitochondria function. These results show that spaceflight as well as altered gravity produced by clinostat rotation affects microtubule and mitochondria organization and results in increases in apoptosis. Grant numbers: NAG 10-0224, NAG2-985.

cDNA microarray reveals altered cytoskeletal gene expression in space-flown leukemic T lymphocytes (Jurkat)

Cytoskeletal disruption and growth arrest consistently occur in space‐flown human acute leukemic T cells (Jurkat). Although the microtubules appear to reorganize during spaceflight, cells remain nonproliferative. To test the hypothesis that spaceflight alters cytoskeletal gene expression and may thus affect cytoskeletal function, we flew Jurkat cells on Space Transportation System (STS) 95 and compared RNA message by cDNA microarray in space‐flown vs. ground controls at 24 h (4,324 genes) and 48 h (>20,000 genes). Messages for 11 cytoskeleton‐related genes, including calponin, dynactin, tropomodulin, keratin 8, two myosins, an ankyrin EST, an actinlike protein, the cytoskeletal linker (plectin), and a centriole‐associated protein (C‐NAP1), were up‐regulated in space‐flown compared with ground control cells; gelsolin precursor was down‐regulated. Up‐regulation of plectin and C‐NAP1 message in both space‐flown cells and vibrated controls is a novel finding and implies their role in vibration damage repair. This first report of cDNA microarray screening of gene expression in space‐flown leukemic T cells also identifies differential expression of genes that regulate growth, metabolism, signal transduction, adhesion, transcription, apoptosis, and tumor suppression. Based on differential expression of cytoskeletal genes, we conclude that centriole‐centriole, membrane‐cytoskeletal, and cytoskeletal filament associations are altered in the orbital phase of spaceflight.

Cytokine secretion by immune cells in space

Cultured, bone marrow–derived macrophages, murine spleen and lymph node cells, and human lymphocytes were tested for their ability to secrete cytokines in space. Lipopolysaccharide‐activated bone marrow macrophages were found to secrete significantly more interleukin‐1 and tumor necrosis factor when stimulated in space than when stimulated on earth. Murine spleen cells stimulated with poly I:C in space released significantly more interferon‐α at 1 and 14 hours after stimulation than cells stimulated on earth. Similarly, murine lymph node T cells and human peripheral blood lymphocytes, stimulated with concanavalin A in space, secreted significantly more interferon‐γ than ground controls. These data suggest that space flight has a significant enhancing effect on immune cell release of cytokines in vitro.

The kinetics of translocation and cellular quantity of protein kinase C in human leukocytes are modified during spaceflight

Protein kinase C (PKC) is a family of serine/threonine kinases that play an important role in mediating intracellular signal transduction in eukaryotes. U937 cells were exposed to microgravity during a space shuttle flight and stimulated with a radiolabeled phorbol ester ([3H]PDBu) to both specifically label and activate translocation of PKC from the cytosol to the particulate fraction of the cell. Although significant translocation of PKC occurred at all g levels, the kinetics of translocation in flight were significantly different from those on the ground. In addition, the total quantity of [3H]PDBu binding PKC was increased in flight compared to cells at 1 g on the ground, whereas the quantity in hypergravity (1.4 g) was decreased with respect to 1 g. Similarly, in purified human peripheral blood T cells the quantity of PKCδ varied in inverse proportion to the g level for some experimental treatments. In addition to these novel findings, the results confirm earlier studies which showed that PKC is sensitive to changes in gravitational acceleration. The mechanisms of cellular gravisensitivity are poorly understood but the demonstrated sensitivity of PKC to this stimulus provides us with a useful means of measuring the effect of altered gravitylevels on early cell activation events.—Hatton, J. P., Gaubert, F., Lewis, M. L., Darsel, Y., Ohlmann, P., Cazenave, J.‐P., Schmitt, D. The kinetics of translocation and cellular quantity of protein kinase C in human leukocytes are modified during spaceflight. FASEB J. 13 (Suppl.), S23–S33 (1999)

Regulation of heat shock protein message in Jurkat cells cultured under serum‐starved and gravity‐altered conditions

Although our understanding of effects of space flight on human physiology has advanced significantly over the past four decades, the potential contribution of stress at the cellular and gene regulation level is not characterized. The objective of this ground‐based study was to evaluate stress gene regulation in cells exposed to altered gravity and environmentally suboptimal conditions. We designed primers to detect message for both the constitutive and inducible forms of the heat shock protein, HSP‐70. Applying the reverse transcriptase‐polymerase chain reaction (RT‐PCR), we probed for HSP‐70 message in human acute T‐cell leukemia cells, Jurkat, subjected to three types of environmental stressors: (1) altered gravity achieved by centrifugation (hypergravity) and randomization of the gravity vector in rotating bioreactors, (2) serum starvation by culture in medium containing 0.05% serum, and (3) temperature elevation (42°C). Temperature elevation, as the positive control, significantly increased HSP‐70 message, while centrifugation and culture in rotating bioreactors did not upregulate heat shock gene expression. We found a fourfold increase in heat shock message in serum‐starved cells. Message for the housekeeping genes, actin and cyclophilin, were constant and comparable to unstressed controls for all treatments. We conclude that gravitational perturbations incurred by centrifugal forces, exceeding those characteristic of a Space Shuttle launch (3g), and culture in rotating bioreactors do not upregulate HSP‐70 gene expression. In addition, we found RT‐PCR useful for evaluating stress in cultured cells. J. Cell. Biochem. 77:127–134, 2000.

Production and action of cytokines in space

B6MP102 cells, a continuously cultured murine bone marrow macrophage cell line, were tested for secretion of tumor necrosis factor-alpha and Interleukin-1 during space flight. We found that B6MP102 cells secreted more tumor necrosis factor-alpha and interleukin-1 when stimulated in space with lipopolysaccharide than controls similarly stimulated on earth. This compared to increased secretion of interferon-beta and -gamma by lymphocytes that was measured on the same shuttle flights. Although space flight enhanced B6MP102 secretion of tumor necrosis factor-alpha, an experiment on a subsequent space flight (STS-50) found that cellular cytotoxicity, mediated by tumor necrosis factor-alpha, was inhibited.

Fas/APO-1 protein is increased in spaceflown lymphocytes (Jurkat)

Human lymphocytes flown on the Space Shuttle respond poorly to mitogen stimulation and populations of the lymphoblastoid T cell line, Jurkat, manifest growth arrest, increase in apoptosis and time- and microgravity-dependent increases in the soluble form of the cell death factor, Fas/APO-1 (sFas). The potential role of apoptosis in population dynamics of space-flown lymphocytes has not been investigated previously. We flew Jurkat cells on Space Transportation System (STS)-80 and STS-95 to determine whether apoptosis and the apparent microgravity-related release of sFas are characteristic of lymphocytes in microgravity. The effects of spaceflight and ground-based tests simulating spaceflight experimental conditions, including high cell density and low serum concentration, were assessed. Immunofluorescence microscopy showed increased cell associated Fas in flown cells. Results of STS-80 and STS-95 confirmed increase in apoptosis during spaceflight and the release of sFas as a repeatable, time-dependent and microgravity-related response. Ground-based tests showed that holding cells at 1.5 million/ml in medium containing 2% serum before launch did not increase sFas. Reports of increased Fas in cells of the elderly and the increases in spaceflown cells suggest possible similarities between aging and spaceflight effects on lymphocytes.

The cytoskeleton, apoptosis, and gene expression in T lymphocytes and other mammalian cells exposed to altered gravity.

Publisher Summary Lymphocytes exposed to spaceflight or altered gravity exhibit significant growth attenuation and changes in metabolism, cytoskeletal structure, and gene expression. A significant body of evidence is accumulating to implicate the cytoskeleton in the atypical response of lymphocytes in microgravity. This chapter reviews the effects of spaceflight and altered gravity on cytoskeletal morphology and expression of cytoskeletal genes in T lymphocytes and established T-cell lines. Other mammalian cell types are included for comparison. The effects of simulated shuttle launch vibration are discussed based on research showing that vibration during launch may disrupt the cytoskeleton and alter gene expression. Ground-based vibration studies provide baseline information for distinguishing effects of vibration from effects of the microgravity environment per se. As cytoskeletal disruption is one of several factors known to exacerbate apoptosis, and because of the apparent increase in programmed cell death in space-flown cells, apoptosis is also discussed in the chapter.

Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat).

Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription‐polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space‐flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up‐regulate the mRNA for hsp70, although the gene encoding hsp27 is up‐regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up‐regulation of hsp27 gene expression in microgravity is a new finding.