Reversion of virus N-1 resistant mutant of blue-green algaNostoc muscorum

Summary The reversion of N-1 virus resistant strain of the algaNostoc muscorum was studied by inoculating parent virus in the resistant culture at various incubations. A fraction of virus resistant cells reverted to wild sensitive type with the reversion rate of 3.99×10^–6/cell/generation.We gratefully acknowledge Dr H.K. Pande and Dr S. Patnaik, Crops and Soils Division.     

Inhibition of protein deacetylation by trichostatin A impairs microtubule-kinetochore attachment

Inhibition of protein deacetylation arrests cells in mitosis, but the mechanism is unknown. To understand why inhibiting protein deacetylation causes cell cycle arrest, we treated HeLa cells beyond G1/S transition with trichostatin A (TSA), a potent protein deacetylase inhibitor, and found that the cells arrested at prometaphase with ectopic spindles and unaligned chromosomes. The hyper-acetylated cells encountered a serious microtubule (MT)-kinetochore attachment problem, although the kinetochores are intact at ultrastructural level. By immunofluorescence staining of kinetochore proteins, we found that the pericentromeric H3K9Me3-HP1 pathway was disrupted and that the CENP-A-dependent outer plate protein dynamics of kinetochores was greatly diminished by the drug treatment. The treatment also caused the loss of chromosome passenger complex (CPC), the proposed error checking system, from centromere and impaired the microtubule dynamics of the cells. Overall, we propose that deacetylation inhibition impairs MT-kinetochore attachment through disrupting the centromere function and altering the kinetochore composition and MT dynamics. Received 30 April 2008; received after revision 28 July 2008; accepted 14 August 2008     

Establishment of normal diploid and malignant heteroploid cell lines from non-treated and benzo(a)pyrene treated hamster embryo cell cultures

Summary Two normal diploid control cell lines and a heteroploid malignant transformed cell line from B(a) P treated hamster embryo cell cultures were established. The 14-month-old B(a)P transformed cell line grew 8-times faster than the 20-month-old control cell line. The control cell line showed normal diploid chromosome complement in 93% cells and heteroploidy in 7% cells while B(a)P treated line showed 83% heteroploid cells and only 17% diploid cells. This is the first report on the establishment of diploid hamster cell cultures grown for extended period. Acknowledgments. This work was supported by the French Ministry of Quality of Life, by the Institut National de la Santé et Recherche Médicale, by the S.E.I.T.A. and by Contract No. 12-14-7001-297 of the Agricultural Research Service, U.S. Dept. of Agriculture, Administered by the Athens, Georgia Area, Richard B. Russell Agricultural Research Center, Athens, Georgia 30604, USA. We thank Mr.B. R. Sharma, Mr.E. J. Radin, Dr.R. Hakim and Dr.Ved Brat for their help during this work.     

Immunocytochemical and enzymatic detection of lysozyme in human colon carcinoma cell lines

Summary Six of a total of 14 human colon carcinoma cell lines produce and secrete lysozyme in vitro. Three also produce the enzyme when propagated in vivo in athymic mice. None of the lysozyme positive cells stained in a manner typical of Paneth cells. Additionally, lysozymes from all six colon lines possess identical molecular weights (14,000 daltons).This work was supported by funds from the Monsanto Company under agreements with Harvard University. We are indebted to A. Ehrlich, S. Kane, and D. Sleeman for excellent technical assistance and to Dr B. Vallee for continued advice and support.J.W.F. and E.M.A. are also members of the Department of Pathology, Harvard Medical School.     

A synergistic factor of an insect granulosis virus agglutinates insect cells

Summary The synergistic factor (SF), a lipoprotein which enhances in vivo and in vitro baculovirus infections, occurs in the matrix of the occlusion body (capsule) of a granulosis virus of the armyworm,Pseudaletia unipuncta. The SF attaches to certain areas on the surfaces of cell plasma membranes of cultured insect cells, resulting in marked cellular agglutination. The minimal amount of SF detectable by agglutination is approximately 3 g/ml. Cultured cells of 6 out of 10 insect species in 3 orders are agglutinated by the SF, but not those of 2 mammalian species and the erythrocytes of 8 vertebrate species.We thank Dr M. Nagata and Dr T. Yamamoto for preparing the synergistic factor and R.T. Hess and E.M. Omi for technical assistance. We also thank the donors who provided the cell lines. This work was supported by the National Science Foundation under Grant No. PCM-8201247.     

Response of an undifferentiated and nullipotential cell line derived from an A/Heston mouse teratocarcinoma to infection with type C ecotropic murine viruses]

The PCC6 cell line, derived from an A/Heston Mouse Teratocarcinoma, and composed of nullipotential embryonic carcinoma cells (ECC), is completely resistant to infection with type C murine ecotropic viruses. It reacts as other cell lines previously studied which are derived from a 129 Mouse teratocarcinoma and composed of multipotential ECC.     

Interferon-alpha and transforming growth factor-β co-induce growth inhibition of human tumor cells

A hallmark of resistance to type I interferons (IFNs) is the lack of antiproliferative responses. We show here that costimulation with IFN-alpha and transforming growth factor beta-1 (TGF-beta) potentiates antiproliferative activity in a sensitive (ME15) and resistant (D10) human melanoma cell line. A DNA microarray-based search for proliferation control genes involved that are cooperatively activated by IFN-alpha and TGF-beta, yielded 28 genes. Among these are the insulin-like growth factor-binding protein 3 (IGFBP3) and the calcium-binding protein S100A2; we demonstrate, that recombinant IGFBP3 protein is a potent growth inhibitor requiring TGF-beta activity. The antiproliferative activity of S100A2 is significantly enhanced by IFN-alpha in stably transfected ME15 or D10 cell lines. We show for the first time that IFN-alpha is a potent inducer of intracellular calcium release required for activation of S100A2. Our study provides a functional link between IFN-alpha and TGF-beta signaling and extends the function of IFN signaling to calcium-sensitive processes.     

Regulation of long-distance transport of mitochondria along microtubules

Mitochondria are cellular organelles of crucial importance, playing roles in cellular life and death. In certain cell types, such as neurons, mitochondria must travel long distances so as to meet metabolic demands of the cell. Mitochondrial movement is essentially microtubule (MT) based and is executed by two main motor proteins, Dynein and Kinesin. The organization of the cellular MT network and the identity of motors dictate mitochondrial transport. Tight coupling between MTs, motors, and the mitochondria is needed for the organelle precise localization. Two adaptor proteins are involved directly in mitochondria-motor coupling, namely Milton known also as TRAK, which is the motor adaptor, and Miro, which is the mitochondrial protein. Here, we discuss the active mitochondria transport process, as well as motor–mitochondria coupling in the context of MT organization in different cell types. We focus on mitochondrial trafficking in different cell types, specifically neurons, migrating cells, and polarized epithelial cells.     

Demonstration of the presence of M-creatine kinase in mammalian myogenic cell lines

Summary Unequivocal identification of M-CK in cell extracts from fused cells of myogenic cell lines is difficult due to almost identical behaviour of the M-CK and a contaminating enzyme activity in electrophoresis. If CK dimers present in cell extracts were subjected to dissociation and reassociation in the presence of exogenous B-CK subunits, the formation of easily identifiable MB-CK was demonstrated, indicating the presence of M-CK in the myogenic rat cell lines.Acknowledgments. We are grateful to Mrs M. Siegrist and Dr D. Turner for communication of preliminary results and helpful discussions, Mrs E.R. Perriard for skillful technical help. Supported by a grant of the Muscular Dystrophy Association Inc. to H.M.E.     

Selection for resistance toBacillus thuringiensis δ-endotoxin in an insect cell line (Choristoneura fumiferana)

Summary A cell line of the spruce budworm (FPMI-CF1) consists of a mixed population of cells that possess variable sensitivity to -endotoxin from crystals ofBacillus thuringiensis. A cell strain was selected from FPMI-CF1 which was resistant to the entomocidal protein extracted fromB. thuringiensis crystals. The resistant character was unstable, however, and could not be maintained in the absence of toxin during growth.The author wishes to thank S.S. Sohi (Forest Pest Management Institute, Canadian Forestry Service, Sault Ste. Marie, Ontario, Canada) for theC.fumiferana cell line, and L.I. Davidson (USDA) for his able technical assistance.     

Comparative studies on the covalent binding of the carcinogen benzo(a)pyrene to DNA in various model systems

Summary The covalent binding of tritiated benzo(a) pyrene (BP) to DNA has been determined in rat liver in vivo, in rat liver perfused in situ, after incubation of BP with liver single cells, with liver homogenate, with liver microsomes and DNA, with fibroblasts from a rat granuloma pouch, and with 2 cell lines. Liver single cells were found to be a valuable compromise between the most sensitive system (microsomal incubation of BP with DNA) and the biologically most relevant system (in vivo).Presented in part at the 10th Annual Meeting of the Union of Swiss Societies of Experimental Biology, Experientia34, 925 (1978), abstract.Acknowledgment. We thank Dr G. Kistler, Institute of Anatomy, University of Zurich, for generously supplying the SIRC and VERO cell lines, and Mr P. Manser for the preparation of the granuloma pouch fibroblasts.     

Cortisol resistant RPMI-1788 lymphocytes become sensitive to cortisol subsequent to a 24-h incubation period in medium containing purified human transcortin

Summary RPMI-1788 lymphocytes (a human cell line) are resistant to cortisol in vitro. Prior incubation for a minimum of 24 h a medium which contains purified human transcortin at a concentration of 50 g/ml renders these cells sensitive to the inhibitory action of cortisol as regards the synthesis of DNA. Only the transcortin-exposed cells contain a cortisol binding species whose sedimentation behavior in a sucrose gradient is identical to that of transcortin.This work was supported in part by grant ET-47 from the American Cancer Society. I thank Mr D. Pina and Dr R. Khaund for their excellent technical assistance.     

Metallothionein promotes regenerative axonal sprouting of dorsal root ganglion neurons after physical axotomy

Prior studies have reported that metallothionein I/II (MT) promote regenerative axonal sprouting and neurite elongation of a variety of central nervous system neurons after injury. In this study, we evaluated whether MT is capable of modulating regenerative axon outgrowth of neurons from the peripheral nervous system. The effect of MT was firstly investigated in dorsal root ganglion (DRG) explants, where axons were scratch-injured in the presence or absence of exogenous MT. The application of MT led to a significant increase in regenerative sprouting of neurons 16 h after injury. We show that the pro-regenerative effect of MT involves an interaction with the low-density lipoprotein receptor megalin, which could be blocked using the competitive antagonist RAP. Pre-treatment with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 also completely abrogated the effect of exogenous MT in promoting axonal outgrowth. Interestingly, we only observed megalin expression in neuronal soma and not axons in the DRG explants. To investigate this matter, an in vitro injury model was established using Campenot chambers, which allowed the application of MT selectively into either the axonal or cell body compartments after scratch injury was performed to axons. At 16 h after injury, regenerating axons were significantly longer only when exogenous MT was applied solely to the soma compartment, in accordance with the localized expression of megalin in neuronal cell bodies. This study provides a clear indication that MT promotes axonal regeneration of DRG neurons, via a megalin- and MAPK-dependent mechanism.     

Proteasome inhibition overcomes the resistance of renal cell carcinoma cells against the PPARγ ligand troglitazone

In order to analyze the effects of peroxisome proliferator-activated receptor-γ (PPARγ) activation on renal cell carcinomas we utilized several cell lines that were treated with the high affinity PPARγ agonist, troglitazone. Incubation of RCC cells with troglitazone resulted in reduced secretion of growth factors that was due to the inhibition of MAP kinase signaling and reduced nuclear localized expression of relB and HIF1alpha. Interestingly, the cell lines used showed a different sensitivity towards apoptosis induction that did not correlate with the inhibition of growth factors or expression of pro- and antiapoptotic molecules. To overcome this resistance the cells were treated with a combination of troglitazone and the proteasome inhibitor, bortezomib. The combination of both compounds induced apoptosis even in cells resistant to both agents alone, due to increased induction of ER-stress and caspase-3 mediated cell death. Received 03 September 2009; received after revision 02 February 2009; accepted 10 February 2009     

Analysis of OCT4 expression in an extended panel of human tumor cell lines from multiple entities and in human mesenchymal stem cells

OCT4 is considered a main regulator of embryonic stem cell pluripotency and self renewal capacity. It was shown that relevant OCT4 expression only occurs in cells of embryonic pluripotent nature. However, several recent publications claimed to have demonstrated OCT4 expression in human somatic tumor cells, human adult stem or progenitor cells and differentiated cells.We analysed 42 human tumor cell lines from 13 entities and human bone marrowderived mesenchymal stem cells (MSC). To validate OCT4 expression we used germ cell tumor (GCT) cell lines, derived xenografts and GCT samples. Analysis by RT-PCR, western blotting, immunocytochemistry and immunohistochemistry was performed. With exception of typical embryonal carcinoma cells, we did not observe reliable OCT4 expression in somatic tumor cell lines and MSC. We suggest that a high level of expression of the OCT4 protein together with its nuclear localization still remains a reliable and definitive feature of cells with embryonic pluripotent nature. Received 30 September 2008; received after revision 05 November 2008; accepted 10 November 2008     

Protein aggregation as primary and characteristic cell reaction to various stresses

Ehrlich carcinoma and EL-4 thymoma ascites cells were subjected in vitro to heat shock, ATP depletion, oxidative stress, Ca²⁺ overlading and iodoacetamide treatment. After the transient stresses, Triton (X-100)-insoluble TIS) fractions were isolated from the cells and analysed by electrophoresis and immunoblotting. All stresses used caused rapid aggregation of cell proteins. This was manifested in a signficant rise in protein content in the TIS fractions. The protein increase was mostly due to and increase in the insolubility of actin, 57 kDa protein of intermediate filaments, 70 kDa heat shock protein (HSP 70), and some specific proteins whose insolubilization was a characteristic sign for each type of cell injury. Different survival rates in the cell lines after either stress corrlated well with differences in their TIS protein accretion. Possible mechanisms for stress-induced protein aggregation and its relationship with cell viability are suggested.     

Understanding microtubule dynamics for improved cancer therapy

Microtubules (MTs), key components of the cytoskeleton, are dynamic polymers of tubulin that form a well-organized network of polarized tube filaments. MT dynamics are highly regulated both spacially and temporally by several MT-related proteins, themselves regulated by several kinases and phosphatases via signaling cascades, and also by coordinated interactions with actin cytoskeleton and adhesion sites. Regulation of MT dynamics is crucial for mitosis, cell migration, cell signaling and trafficking. MT-targeted drugs (MTDs), which constitute a major anticancer drug family with antimitotic and antiangiogenic properties, inhibit tumor progression mainly by altering MT dynamics in both cancer and endothelial cells. Identification of proteins regulating the MT network will lead to a better understanding of tumor progression regulators and will be helpful in improving cancer therapy. Received 22 July 2005; received after revision 8 September 2005; accepted 12 September 2005     

Narciclasine,an inhibitor of protein synthesis. Action onAllium cepa L. root meristems

Summary Shortening of nucleologenesis time in a synchronous cell population, labelled as binucleate, by a caffeine pulse and fall in frequency of prophases are related to narciclasine inhibition of protein synthesis inAllium cepa L. meristems.Acknowledgments. We gratefully acknowledge Dr D. Vázquez and Dr A. Jiménez, from the Instituto de Bioquímica de Macromoléculas, for calling our attention to this protein synthesis inhibitor and providing us samples both of narciclasine andNarcissus bulbs. We also greatly appreciate Dr B. Rodriguez and Dr F. M. Panizo of the Instituto de Química Orgánica (Departamento de Productos Naturales) for their valuable help in isolation and characterization of narciclasine, and for laboratory facilities. The work has been partially supported by the Comisión Asesora para la Investigación Científica (Spain). A.S. has a fellowship awarded by the Instituto de Cultura Hispánica, Spain.     

TRAIL induces proliferation of human glioma cells by c-FLIPL-mediated activation of ERK1/2

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in TRAIL-sensitive human malignant glioma cells. We show for the first time that TRAIL stimulates cell growth in TRAIL-resistant glioma cells. TRAIL-induced cell growth in resistant cells occurred through increased cell cycle progression as determined by flow cytometry and Western blot analysis of retinoblastoma protein phosphorylation. Western blot analysis of TRAIL-treated resistant cells revealed phosphorylation of ERK1/2 proteins and in vitro kinase analysis confirmed the activation of the ERK1/2 kinases. Inhibition of MEK1 eliminated both TRAIL-induced ERK1/2 activation and cell proliferation. In addition, siRNA inhibition of c-FLIP expression eliminates TRAIL-induced ERK1/2 activation and proliferation. Furthermore, overexpression of c-FLIP_L potentiates TRAIL-induced ERK1/2 activation and proliferation of resistant glioma cells. Our results have shown for the first time that TRAIL-induced ERK1/2 activation and proliferation of TRAIL-resistant human glioma cells is dependent upon the expression of the long form of the caspase-8 inhibitor c-FLIP_L. Received 2 November 2007; received after revision 14 December 2007; accepted 21 December 2007     

Selection of cell lines resistant to cyclic AMP and theophylline from murine plasmocytoma MOPC 173. Cross resistance to ouabain and concanavalin A]

Using cAMP or theophyllin as selective agents, we obtained from contact inhibited and non contact inhibited cell lines, derived from the same plasmocytoma, different resistant cell lines. All of them were cAMP and theophyllin resistant and also ouabain and con A resistant as judged by the growth curves in presence of the different drugs.