cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G1 phase of the cell cycle

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G₁ phase of the cell cycle instead of G₀. Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G₀ phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G₁, 12% were in S and 37% in G₂ phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased (P <0.01) [³H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more (P <0.01) [³H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G₁ phase of the cycle to enhance IGF-I effects in cell proliferation.     

EFFECT OF METABOLIC INHIBITORS ON NUCLEAR PORE FORMATION DURING THE HELA S3 CELL CYCLE

The effect of various antimetabolites on nuclear pore formation was studied in synchronized HeLa S₃ cells. The nuclear size was determined by light microscopy and the pore number per unit area of nuclear surface by the freeze-etching technique and electron microscopy. It was found that the inhibition of DNA replication or ribosomal RNA synthesis has no effect on nuclear size increase or pore formation. However, the inhibition of ATP synthesis effectively stops nuclear pore formation. Cycloheximide blocks nuclear pore formation at the same time during G₁ phase of the cell cycle when nuclear size increase is blocked by high concentrations of actinomycin D. This suggests that certain proteins or other factors leading to pore formation and nuclear size increase are transcribed and synthesized at about 3–4 h after mitosis, i.e., about 1–2 h before S phase begins.     

Protein synthesis during the cell cycle of L5178Y cells

There are two peaks of ³H-leucine incorporation in the cell cycle of L5178Y cells. The first, during S stage, corresponds to a peak of ³H-leucine incorporation into the nuclear fraction. The second, during S or early G2, corresponds to a peak of ³H-leucine incorporation into the mitochondrial fraction. The rate of protein synthesis is unique for the proteins from each of the four fractions, nuclear, mitochondrial, microsomal, and soluble.The SDS polyacrylamide-gel electrophoretic patterns of ³H-leucine incorporation were different among three subcellular fractions: nuclear, mitochondrial, and microsomal + soluble. However, the incorporation pattern for each fraction remains qualitatively the same throughout the cell cycle.     

The use of zonal centrifugation to study membrane formation during the life cycle of mammalian cells. Synthesis of `marker'' enzymes and other components of cellular organelles

1. The activity of enzymes characteristic of microsomes (NADPH-cytochrome c reductase and uridine diphosphatase) and of inner mitochondrial membranes (cytochrome c oxidase and succinate-cytochrome c reductase) increases during the cell cycle of P815Y neoplastic mast cells in concert with total protein. The activity of glutamate dehydrogenase, an enzyme of the mitochondrial matrix, increases in a somewhat different manner. 2. The specific activity of mitochondrial structures involved in energy-coupling measured with a fluorescent probe remains constant during the cell cycle. 3. Mitochondrial and microsomal protein increases during the cycle at the same time as total protein; nuclear protein increases rather more sharply. 4. The rate of incorporation of labelled choline or inositol into nuclear, mitochondrial or microsomal phospholipid during the cell cycle follows the rate of incorporation into total phospholipid. 5. It is concluded that the major components of cellular membranes are synthesized, like total protein or phospholipid, throughout most of the intermitotic period.     

Oncoprotein 18 levels and phosphorylation mediate megakaryocyte polyploidization in human erythroleukemia cells

Megakaryocytes undergo an unusual cell cycle during differentiation that results in polyploidy through largely unknown mechanism(s). It has been shown that serine phosphorylation of oncoprotein 18 (Op18) is required for cell cycle progression specifically at the G2/M transition. Moreover, mutant forms of Op18 that are defective in one or more of the four serine residues induce G2/M arrest and subsequent polyploidization. Op18 phosphorylation is rapidly induced with phorbol myristate acetate (PMA) treatment in a wide range of human cells. In this study, we investigated the role of Op18 in PMA induced polyploidization during megakaryocyte differentiation of the human erythroleukemia cell line. Crucial to the molecular analysis of megakaryocyte differentiation, is the ability to fractionate cell populations with different ploidy levels. We have utilized cell elutriation as a fractionation strategy to analyze Op18 expression in synchronized cell subpopulations in different phases of the cell cycle or with progressive megakaryocyte polyploidization. In the absence of PMA, increased phosphorylation of Op18 was observed in HEL cells during cell cycle progression, as for other proliferating cells. However, in contrast to Jurkat leukemia cells chosen as control, HEL cells exhibited a lack of Op18 phosphorylation in response to PMA, which was accompanied by polyploidization and differentiation along the megakaryocytic lineage. To further determine the role of Op18 in polyploidization, HEL cells were transfected with different Op18 expression constructs. Differences in cell survival and polyploidization were observed between high and low Op18 expressors. An increased Op18 level reduced cell survival during the early stage of PMA induced megakaryocyte differentiation, but enhanced polyploidization efficiency. Our findings suggest that maintenance of a high level of unphosphorylated Op18 is required for efficient polyploidization during the differentiation program of megakaryocytes.     

Cell Cycle Activity and β-Tubulin Accumulation During Dormancy Breaking of Acer platanoides L. seeds

Cell cycle events in embryo axes of Norway maple (Acer platanoides L.) seeds were studied during dormancy breaking by flow cytometric analyses of the nuclear DNA content and by immunodetection of β-tubulin. Most embryonic nuclei of dry, fully matured seeds were arrested in the G₂ phase of the cell cycle. In addition, the lowest content of β-tubulin was detected in dry, mature seeds. Imbibition in water and cold stratification resulted in a decrease in the number of nuclei in G₂, and a simultaneous increase in β-tubulin content. In germinated seeds the content of β-tubulin was the highest and the number of cells in G₂ was the lowest. Both cell cycle events preceded cell expansion and division and subsequent growth of the radicle through the seed coat. The anatomical investigation has proved that the main reason for decrease in the number of nuclei in G₂ is mitosis, started with seeds germination (radicle protrusion). The activation of the cell cycle and the β-tubulin accumulation were associated with embryo dormancy breaking. This revised version was published online in July 2006 with corrections to the Cover Date.     

Splenic lymphocytes of adult Xenopus respond differentially to PMA in vitro by either dying or dividing: significance for cancer resistance in this species

Wild-type populations of amphibians, unlike mammalians, appear to be resistant to spontaneous and chemically induced neoplasms. Few true cancers have been reported for non-isogeneic members of Xenopus laevis, despite their widespread use in laboratories around the world. Injection of even the most powerful direct mammalian oncogens e.g. N-methyl N-nitrosourea, that depleted specific populations of T lymphocytes, did not induce cancer. Phorbol diesters, e.g. PMA, are mitogens and apoptogens in both amphibian, and mammalian immunocytes. In mammalian cells, regulation of the cell cycle and of apoptosis are often intimately linked, however, a disjunction in time between early apoptosis and later cell cycling, has been observed with PMA-treated Xenopus splenocytes. Thus, a particular difference between amphibians and mammals may be the requirement to enter the cell cycle before a progression to death by apoptosis. This hypothesis was tested here using dual staining flow cytometry. Xenopus laevis splenocytes were cultured for 8, 24 and 48 hours with phorbol 12-myristate 13-acetate (PMA), previously shown to be mitogenic and apoptotic with mature Xenopus lymphocytes. The cells were stained with FITC-conjugated Annexin V or with FITC-labeled deoxyuridine triphosphates (FITC-dUTP) to assay for the apoptotic markers phosphotidylserine or DNA strand breaks respectively. Phycoerythrin (PE)-conjugated anti-human proliferating cell nuclear antigen (PE-PCNA) was used as a cell cycle marker that is present during the entire cell cycle. Propidium iodide (PI) binds DNA and was used to assay for late stage apoptosis, as well as to assess DNA content.Significantly higher levels of apoptosis develop rapidly in PMA-exposed splenocytes and are maintained at 24 hours, declining by 48 hours. Cells expressing PCNA or incorporating PI in excess of the normal genomic level were found by 48 hours following PMA exposure. The absence of any significant rise in a small (<5%) dual staining cell population indicates that the apoptotic cell population remained distinct from cells already in the cell cycle from the onset of PMA exposure. Thus, Xenopus splenocytes respond differentially to PMA. Those that undergo apoptosis rapidly were quiescent, non-cycling small lymphocytes. Moreover, the cells that eventually begin division, following PMA exposure, were unaffected by the early apoptois and do not themselves die while in the cell cycle. The rapid apoptotic response of X. laevis cells to PMA may confer a natural cancer resistance in this species, as cells that fail to enter the cell cycle after exposure to cancer promoting reagents cannot express genetic destabilization that might have led to transformation.     

Glycoside and Polysaccharide Hydrolase Activity of the Rumen Anaerobic Fungus Caecomyces communis (Sphaeromonas communis SENSU ORPIN) at Early and Final Stages of the Developmental Cycle

The rumen anaerobic fungus Caecomyces communis was grown in a fermentor in Lowe medium. We studied four polysaccharide hydrolases and three glycoside hydrolases at early and final stages. We found a difference in cell association for these enzymes depending on the developmental stage. The endocellulase and β-D-fucosidase were early synthesized, and their activities decreased at the end of the developmental cycle. On the contrary, the β-D-glucosidase, β-D-xylosidase and xylanase activities increased during the cycle. The avicelase and the CM-cellulase activities linked with thalli increased, whereas the extracellular activities of these enzymes decreased.     

HTm4基因在造血细胞周期调控中的作用

为了研究HTm4基因在造血细胞细胞周期调控中的作用,以佛波酯(phorbol 12-myristate 13-acetate,PMA)诱导K562细胞分化为模型,利用流式细胞术(FACS)及半定量RT-PCR对分化过程中细胞周期的变化及HTm4基因的表达进行了分析,并利用Tet-Off调控表达系统,将HTm4基因以及C端功能域缺失的HTm4-ct转染K562细胞,观察对细胞周期的影响。结果表明,PMA同时引起了K562细胞的增殖和分化,G0／G1期细胞的比例以及HTm4基因的表达均呈现出波浪形的变化趋势,说明HTm4基因可能参与了细胞退出细胞周期的过程。HTm4基因转染后引起K562细胞滞留于G0／G1期,但C端功能域缺失的HTm4-ct没有此作用,说明C端功能域在HTm4基因调控细胞周期的功能中发挥重要作用。     

The retinoblastoma protein is phosphorylated during specific phases of the cell cycle

p105-RB is the product of the retinoblastoma tumor suppressor gene. It is a nuclear phosphoprotein hypothesized to act as an inhibitor of cellular proliferation, yet surprisingly it is present in actively dividing cells. To look for changes in p105-RB that may regulate its activity during the cell cycle, we generated synchronized cell populations and followed their progression through the cell cycle. p105-RB is synthesized throughout the cycle, but is phosphorylated in a phase-specific manner. In the G0 and G1 phases of the cell cycle, an unphosphorylated species of the protein is the only detectable form, whereas in the S and G2/M phases, multiple phosphorylated species of p105-RB are detected.     

STRUCTURE AND DISTRIBUTION OF GLUCOMANNAN AND SULFATED GLUCAN IN THE CELL WALLS OF THE RED ALGA KAPPAPHYCUS ALVAREZII (GIGARTINALES, RHODOPHYTA)

Two new polysaccharides were isolated from the cell walls of the carrageenan producing red seaweed Kappaphycus alvarezii (Doty) Doty. They were characterized by chemical analyses, enzymatic degradations, and nuclear magnetic resonance spectroscopy. One was a 4.0 M NaOH soluble β-(1,4)- d -glucomannan that mostly precipitated upon neutralization and dialysis. It was composed of about 82 residues, and 70% of its glucose and mannose were released by a commercial cellulase enzyme complex. The disaccharide β- d -Man (1→4) d -Glc was recovered from the hydrolysate during the first hours of degradation and confirmed the chemical structure of the polysaccharide. The other polysaccharide was extracted with 1.5 M NaOH and was identified as a sulfated glucan of degree of polymerization of about 180 1,4-linked β-glucose containing 10% 1,3-linkages. The sulfate was located on C-6 of 64% of the 4-linked glucose residues. A third alkali-soluble polysaccharide rich in galactose was also detected. The distribution of the glucomannan and galactose containing polysaccharides was inversely related to the algal cell size. Potential functions of these alkali-soluble polymers are discussed in the context of cell wall polysaccharide assembly.     

Mitochondrial DNA synthesis in synchronous cultures of the yeast,Saccharomyces cerevisiae

The synthesis of mitochondrial DNA (mtDNA) has been investigated by three independent methods of analysis during consecutive synchronous cell cycles in the yeast, Saccharomyces cerevisiae. The rates of pulse-label incorporation indicate maximal [³H]adenine uptake into mtDNA at the time of nuclear DNA synthesis. In contrast, the relative concentrations of mtDNA as determined by both the ratio of mtDNA to total cellular DNA and by the kinetics of isotope dilution analysis were found to increase continuously during synchronous growth. We conclude that whereas nuclear DNA replicates discontinuously during the cell cycle, mitochondrial DNA is synthesized continuously during this time. The discontinuous pattern of pulse-label incorporation into mtDNA is not considered to reflect its true mode of replication during the cell cycle.     

Synthesis and characterization of nuclear matrix proteins during the cell cycle of Physarum polycephalum

Pulse-labelling with [³⁵S]-methionine/cysteine of macroplasmodia of the myxomycete Physarum polycephalum at different time points of the cell cycle reveals that the majority of nuclear matrix proteins is synthesized and assembled into nuclear structures without a pronounced cell cycle periodicity. Bulk nuclear histones on one hand and nuclear matrix associated histones on the other hand assemble with a different cell cycle periodicity suggesting specific functions of nuclear matrix bound chromatin. Characterization of the nuclear matrix by immunoblotting and immunofluorescence techniques with several antisera against vertebrate lamins shows the existence of lamin-homologous proteins in Physarum.     

Yeast karyopherin Kap95 is required for cell cycle progression at Start

Background  

The control of the subcellular localization of cell cycle regulators has emerged as a crucial mechanism in cell division regulation. The active transport of proteins between the nucleus and the cytoplasm is mediated by the transport receptors of the β-karyopherin family. In this work we characterized the terminal phenotype of a mutant strain in β-karyopherin Kap95, a component of the classical nuclear import pathway.     

Investigation of poly ( β-L-malic acid) production by strains of Aureobasidium pullulans

 Eight strains of the genus Aureobasidium obtained from culture collections were tested for their capability to produce poly(β-L-malic acid) (PMA). Four of the tested strains showed positive results. The most productive strain, A. pullulans CBS 591.75, was used to study the production of PMA in stirred-tank reactors. It was found that PMA was mainly produced in the late exponential phase, and the production related positively to glucose consumption. At the beginning of the fermentation the pH increased from 4.0 to about 7.0; subsequently the pH decreased and remained stable at around 3.0–3.5 for several days. Temperatures higher than 25^°C were detrimental to PMA production and cell growth. PMA production and cell growth at 20^°C and 25^°C exhibited no significant differences. PMA production and cell growth were studied under pH-controlled fermentation (at pH 2.0, 4.0, 5.5). The highest PMA production occurred at pH 4.0. PMA production was reduced at pH 2.0 although quite reasonable cell growth occurred at this pH value. Under optimized conditions 9.8 g PMA/l was produced during 9 days of fermentation in the stirred-tank reactors with an overall yield of 0.11 g PMA/g glucose. A procedure for the isolation of PMA and its separation from the other components of the fermentation broth was developed. The isolated PMA was characterized by ¹H and ¹³C-NMR spectroscopy as well as by infrared absorption spectroscopy. Gel-permeation chromatography revealed a relative molecular mass of approximately 3000–5000 by comparison with polyethylene glycol standards. Received: 13 February 1996/Received revision: 25 April 1996/Accepted: 1 May 1996     

Accessory cell stimulation of T cell proliferation requires active antigen processing, Ia-restricted antigen presentation, and a separate nonspecific 2nd signal

The roles of Ia+ accessory cells in H-2-restricted stimulation of antigen-specific T cell proliferation were explored in an in vitro model. L-glutamic acid60-L-alanine30-L-tyrosine10-(GAT) primed BALB/c nylon wool-passed T cells were depleted of Ia+ antigen-presenting cells (APC) by treatment with monoclonal anti-Ia antibody plus complement. Such cells failed to respond to soluble GAT, or to soluble GAT in the presence of phorbol myristic acetate (PMA), which is known to stimulate production of, or replace, IL-1 in vitro. Addition of gamma-irradiated syngeneic spleen cells reconstituted the response to soluble GAT, but addition of ultraviolet (UV) light-irradiated spleen cells did not, even in the presence of PMA. Preincubation of cells with GAT for 24 hr, followed by washing, then gamma irradiation, generated a cell population able to stimulate GAT-primed T cells to proliferate. The same pulsed cells exposed to UV irradiation failed to stimulate T cell responses unless PMA was added to the cultures. The relevant cells in this UV-irradiated population are Ia+. It is concluded that a finite period of time for interaction of metabolically intact APC with antigen is required before creation of an appropriate (Ia + antigen) signal recognized by the T cell. In addition to such Ia-restricted antigen presentation, however, a 2nd nonspecific signal, again requiring metabolically active APC for elaboration, is necessary for detectable T cell activation. These studies thus define 3 separable activities of APC during the process of H-2 restricted T cell activation.     

Autocrine, paracrine and juxtacrine signaling by EGFR ligands

Receptor and cytoplasmic protein tyrosine kinases play prominent roles in the control of a range of cellular processes during embryonic development and in the regulation of many metabolic and physiological processes in a variety of tissues and organs. The epidermal growth factor receptor (EGFR) is a well-known and versatile signal transducer that has been highly conserved during evolution. It functions in a wide range of cellular processes, including cell fate determination, proliferation, cell migration and apoptosis. The number of ligands that can activate the EGF receptor has increased during evolution. These ligands are synthesized as membrane-anchored precursor forms that are later shed by metalloproteinase-dependent cleavage to generate soluble ligands. In certain circumstances the membrane anchored isoforms as well as soluble growth factors may also act as biologically active ligands; therefore depending on the circumstances these ligands may induce juxtacrine, autocrine, paracrine and/or endocrine signaling. In this review, we discuss the different ways that EGFR ligands can activate the receptor and the possible biological implications.