Inhibition of phosphorylation of cellular dUTP nucleotidohydrolase as a consequence of herpes simplex virus infection

During an infection with herpes simplex virus, activity of cellular dUTPase decreases as a function of time, post-infection, while virus-encoded dUTPase activity increases. Prelabeling of cells with 35S-methionine and immunoprecipitation analysis, using monoclonal antibodies, indicates that cellular dUTPase protein levels remain the same (with respect to levels in uninfected cells) throughout the infection period. New synthesis of cellular dUTPase does not occur in infected cells as determined by 35S-methionine labeling during infection. Further characterization of the cellular dUTPase, in uninfected cells, reveals that the protein is post-translationally phosphorylated at serine residues. Pulse labeling of virus-infected cells with 32P-orthophosphate reveals that the phosphorylation rate of the cellular dUTPase protein decreases significantly as a function of time post-infection. In an effort to establish that phosphate turnover was occurring on the cellular dUTPase protein, cells were prelabeled with 32P-orthophosphate and then infected with HSV in the absence of label. Evidence from this experiment indicates that the phosphate moiety is removed from the cellular dUTPase protein during the infection. A series of viable virus mutants was generated by insertional inactivation of the HSV dUTPase gene. These mutants do not express viral dUTPase activity and HSV dUTPase protein is not detected by western blot analysis. However, in contrast to the wild-type situation, these mutant virus retain significant cellular dUTPase activity throughout infection. Interestingly, phosphorylation of cellular dUTPase protein is now readily detectable in each of the mutant virus-infected cells. These studies indicate that cellular dUTPase activity is diminished in wild-type HSV-infected cells by a process of dephosphorylation. It also appears that in mutant HSV, lacking the virus dUTPase, the mechanism of dephosphorylation and thus inactivation of cellular dUTPase is not functional. The end result is that the mutant virus can now rely on the cellular activity for its survival.     

Involvement of Sib proteins in the regulation of cellular adhesion in Dictyostelium discoideum

Molecular mechanisms ensuring cellular adhesion have been studied in detail in Dictyostelium amoebae, but little is known about the regulation of cellular adhesion in these cells. Here, we show that cellular adhesion is regulated in Dictyostelium, notably by the concentration of a cellular secreted factor accumulating in the medium. This constitutes a quorum-sensing mechanism allowing coordinated regulation of cellular adhesion in a Dictyostelium population. In order to understand the mechanism underlying this regulation, we analyzed the expression of recently identified Dictyostelium adhesion molecules (Sib proteins) that present features also found in mammalian integrins. sibA and sibC are both expressed in vegetative Dictyostelium cells, but the expression of sibC is repressed strongly in conditions where cellular adhesion decreases. Analysis of sibA and sibC mutant cells further suggests that variations in the expression levels of sibC account largely for changes in cellular adhesion in response to environmental cues.     

DNA synthesis in polyoma virus infection. II. Relationship between viral DNA replication and initiation of cellular DNA replicons

The rate of synthesis of cellular DNA is stimulated in stationary phase mouse embryo cells infected with polyoma virus. Nascent cellular DNA strands pulselabeled with [³H]thymidine in the presence of replicating viral DNA are smaller, by an average of 2·1 × 10⁷ daltons, than DNA made under similar conditions in uninfected cells. Previous work (Cheevers et al., 1972) has indicated that this observation is the consequence of activation in infected cells of cellular DNA initiation sites not in operation during a similar pulse-labeling interval in uninfected cells. Similar results were obtained using cells infected with the temperature-sensitive Ts-a mutant of polyoma at 32 °C, which permits both the induction of cellular DNA synthesis and replication of viral DNA. However, at a temperature of 39 °C, which permits only the induction of cellular DNA replication in Ts-a-infected cells, the size of newly synthesized DNA is not different from that of uninfected cells. Similarly, in rat embryo cells abortively infected with polyoma (wild-type), stimulation of cellular DNA synthesis occurs but viral DNA replication is restricted, and no difference is apparent in the size of newly formed DNA as compared to uninfected cells. These results are interpreted to mean that in productively infected cells, polyoma DNA and some regions of the host genome may be co-ordinately replicated.     

Translational control of protein synthesis after infection by vesicular stomatitis virus.

Four hours after infection of BHK cells by vesicular stomatitis virus (VSV), the rate of total protein synthesis was about 65% that of uninfected cells and synthesis of the 12 to 15 predominant cellular polypeptides was reduced to a level about 25% that of control cells. As determined by in vitro translation of isolated RNA and both one- and two-dimensional gel analyses of the products, all predominant cellular mRNA's remained intact and translatable after infection. The total amount of translatable mRNA per cell increased about threefold after infection; this additional mRNA directed synthesis of the five VSV structural proteins. To determine the subcellular localization of cellular and viral mRNA before and after infection, RNA from various sizes of polysomes and nonpolysomal ribonucleoproteins (RNPs) was isolated from infected and noninfected cells and translated in vitro. Over 80% of most predominant species of cellular mRNA was bound to polysomes in control cells, and over 60% was bound in infected cells. Only 2 of the 12 predominant species of translatable cellular mRNA's were localized to the RNP fraction, both in infected and in uninfected cells. The average size of polysomes translating individual cellular mRNA's was reduced about two- to threefold after infection. For example, in uninfected cells, actin (molecular weight 42,000) mRNA was found predominantly on polysomes with 12 ribosomes; after infection it was found on polysomes with five ribosomes, the same size of polysomes that were translating VSV N (molecular weight 52,000) and M (molecular weight 35,000) mRNA. We conclude that the inhibition of cellular protein synthesis after VSV infection is due, in large measure, to competition for ribosomes by a large excess of viral mRNA. The efficiency of initiation of translation on cellular and viral mRNA's is about the same in infected cells; cellular ribosomes are simply distributed among more mRNA's than are present in growing cells. About 20 to 30% of each of the predominant cellular and viral mRNA's were present in RNP particles in infected cells and were presumably inactive in protein synthesis. There was no preferential sequestration of cellular or viral mRNA's in RNPs after infection.     

Multiple receptors mediate apoJ-dependent clearance of cellular debris into nonprofessional phagocytes.

Phagocytosis of apoptotic, senescent, and dying cells by macrophages is a well characterized process. More recently it has been shown that in addition to macrophages vital neighboring cells in the affected tissue participate in the cellular clearance. While scavenger receptors have been shown to mediate uptake into macrophages, it is poorly understood how cellular debris is internalized by nonprofessional phagocytes. We here analyze the endocytic activity of vital fibroblasts and epithelial cells exposed to cellular debris and membrane remnants. We show a mutual stimulation in the endocytosis of debris and apolipoproteinJ (clusterin) in these cells. Experiments using RAP (receptor-associated protein) to block ligand binding to LRP and megalin as well as studies in LRP- and megalin-deficient cells suggest that the uptake of apoJ and cellular debris is mediated by megalin, LRP, and yet unidentified internalization mechanisms.     

Delivering cellular therapies: lessons learned from ex vivo culture and clinical applications of hematopoietic cells

Advances in stem cell biology and cellular therapy have led to promising treatments in a range of incurable diseases. However, it is unclear whether primitive stem cells can be delivered to damage tissue for regeneration of functional mature cells or stem cells must be stimulated to differentiate into mature cells in vitro and these cells delivered to patients. A range of other questions remains to be determined including how to formulate cellular products for in vivo delivery and how to undertake pharmacological testing of cellular products. Insights into these questions can be obtained from hematopoietic stem cells (HSC) which have been used for the past 50 years in bone marrow transplantation for regeneration of blood cells in patients undergoing high dose chemotherapy to treat cancer. The differentiation of HSC into mature blood cells is controlled by proteins called hematopoietic growth factors and these factors have been used to generate cellular products in vitro for clinical applications. This chapter will review some of the results of cellular therapies performed with HSC and the lessons that can be learned from these studies.     

Down-regulation of phosphoglucose isomerase/autocrine motility factor expression sensitizes human fibrosarcoma cells to oxidative stress leading to cellular senescence

Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is a housekeeping gene product present in all cells, is an essential enzyme of catabolic glycolysis and anabolic gluconeogenesis, and regulates tumor cell growth and metastasis. Because glycolytic enzyme up-regulation of expression contributes to glycolytic flux, leading to increased of cell growth and a resistance to cellular stress of normal fibroblasts whereas down-regulation of PGI/AMF leads to mesenchymal-to-epithelial transition in tumor cells, we examined the involvement of PGI/AMF in overcoming cellular senescence in cancer cells. PGI/AMF cellular expression in HT1080 human fibrosarcoma was down-regulated by small interfering RNA methodology, which resulted in an increased sensitivity to oxidative stress and oxidative stress-induced cellular senescence. Signaling analysis revealed that the senescence pathway involving p21 cyclin-dependent kinase inhibitor was up-regulated in PGI/AMF knockdown cells and that superoxide dismutase is the upstream regulator protein of p21-mediated cellular senescence. A specific inhibitor of PGI/AMF induced cellular senescence and p21 expression in tumor cells exposed to an oxidative stress environment. Taken together, the results presented here suggest that PGI/AMF is involved in oxidative stress-induced cellular senescence and should bring novel insights into the control of cellular growth leading to a new methodology for cancer treatment.     

Cellular and molecular basis of skeletal muscle hystogenesis

The molecular basis of development and regeneration of skeletal muscles are reviewed. A model of parent-progeny relationships of mature animals?? skeletal muscles is proposed. Different cellular populations that contribute to myogenesis in vivo and in vitro are described. Both well-known typical cellular sources for muscle regeneration (satellite cells, muscle derived stem cells) and alternative cellular sources (CD133+ cells, pericytes, SP-cells from muscles and from bone marrow, mesangioblasts, embryonic stem cells and mesenchymal stromal cells) are presented. Moreover, some evidence for the existence of ectopic myogenic precursors in nonmuscle tissues is given.     

Calcitriol–copper interaction leads to non enzymatic,reactive oxygen species mediated DNA breakage and modulation of cellular redox scavengers in hepatocellular carcinoma

Calcitriol is the metabolically active form of Vitamin D and is known to kill cancer cells. Using the rat model of DEN induced hepatocellular carcinoma we show that there is a marked increase in cellular levels of copper in hepatocellular carcinoma and that calcitriol–copper interaction leads to reactive oxygen species mediated DNA breakage selectively in hepatocellular carcinoma cells. In vivo studies show that calcitriol selectively induces severe fluctuations in cellular enzymatic and non enzymatic scavengers of reactive oxygen species in the malignant tissue. Lipid peroxidation, a well established marker of oxidative stress, was found to increase, and substantial cellular DNA breakage was observed. We propose that calcitriol is a proxidant in the cellular milieu of hepatocellular carcinoma cells, and this copper mediated prooxidant action of calcitriol causes selective DNA breakage in malignant cells, while sparing normal (non malignant) cells.     

Cellular tumorigenicity in nude mice. Role of susceptibility to natural killer cells

The athymic nude mouse is a useful animal model for assaying the neoplastic growth potential in vivo of animal cells transformed in vitro. Despite the demonstrated absence of thymus-dependent immunological functions, however, the nude mouse has now been shown to reject transplants of certain highly malignant heterologous tumors. In addition, a few transformed mammalian cell lines that exhibit all or most of the cellular phenotypes usually associated with malignancy fail to grow as tumors when injected into nude mice. In a continuing study to identify the in vitro phenotypes associated with tumor-forming ability in vivo, we investigated the role of cellular susceptibility to the naturally occurring, thymus-independent lymphocytes (natural killer or NK cells) in determining tumor induction by animal cells in nude mice. A representative collection of animal cells (ranging from normal human diploid cell strains to highly tumorigenic clonal cell lines, either transformed in vitro or derived from experimental tumors) was tested to see if the ability of cells to form tumors is consistently correlated with their susceptibility to NK cell-mediated lysis measured in vitro with splenic leukocytes from nude mice. If the physiological role of the NK cells in vivo were to recognize, and possibly to destroy, incipient tumor cells in situ, a direct association between cellular tumorigenicity and susceptibility to NK activity, might be expected. If, on the other hand, the formation of growing tumors by animal cells in nude mice depended on their ability to escape the cytolytic activity of NK cells, cellular tumorigenicity would be associated with cellular resistance to NK cells. Results obtained in this study failed to confirm either of these associations. Thus, cellular suscepbibility to NK cells, at least as determined by direct cytotoxicity assay in vitro, is not a useful predictive indicator of cellular tumorigenicity in nude mice.     

The ratio of phosphatidylcholine to phosphatidylethanolamine does not predict integrity of growing MT58 Chinese hamster ovary cells

Phosphatidylcholine (PC) homeostasis is important for maintaining cellular growth and survival. Cellular growth and apoptosis may also be influenced by the PC to phosphatidylethanolamine (PE) ratio as a reduction in this ratio can result in a loss of membrane integrity. To investigate whether a reduced PC:PE ratio influences cellular growth and apoptosis, we utilized the MT58 cell line, which contains a thermo-sensitive mutation in CTP:phosphocholine cytidylyltransferase-α, the rate-limiting enzyme for PC biosynthesis. Incubation of MT58 cells at the restrictive temperature of 41°C results in a reduction of cellular PC and induces apoptosis. Furthermore, MT58 cells have a 50% reduction in the PC:PE ratio when incubated at 41°C. In an attempt to normalize the PC:PE ratio, which may stabilize cellular membranes and rescue MT58 cells from apoptosis, the cells were treated with either silencing RNA to impair PE biosynthesis or lysophosphatidylcholine to increase PC mass. Impairing PE biosynthesis in MT58 cells reduced cellular PE and PC concentrations by 30% and 20%, but did not normalize the PC:PE ratio. Loss of both phospholipids enhanced the onset of apoptosis in MT58 cells. Lysophosphatidylcholine normalized cellular PC, increased PE mass by 10%, restored cellular growth and prevented apoptosis of MT58 cells without normalizing the PC:PE ratio. Furthermore, total amount of cellular PC and PE, but not the PC:PE ratio, correlated with cellular growth (R(2)=0.76), and inversely with cellular apoptosis (R(2)=0.97). These data suggest the total cellular amount of PC and PE, not the PC:PE ratio, influences growth and membrane integrity of MT58 cells.     

Conservation of the basic pattern of cellular amino acid composition during biological evolution in plants

The cellular amino acid composition of plant cells was analyzed. The callus of carrot (Daucus carota), leaves of Torenia fournieri and protocomb-like body of Cymbidium, s.p. were examined as examples of plant cells. The cellular amino acid compositions differed in the plant cells, but their basic patterns were quite similar. It is concluded that the basic pattern of the cellular amino acid composition is conserved in all terrestrial organisms, including plants.     

Scanning electron microscopy of the aggregation of head mesoderm cells from chick embryos

Head mesoderm cells from chick embryos at different stages of development were dissociated and cultured on plastic coverslips. In all cultures several cellular aggregates were described by means of scanning electron microscopy. Isolated cells present filopodia and lamellipodia. However, when mesoderm cells make contact with one another the filopodial and lamellipodial activity in the contact cellular edge disappear. Thus, the cells into cellular clusters do not present projections. The clusters were circular and bidimensional in character. The scanning electron microscopic observations showed that it is the type 1 variant of "contact inhibition of locomotion" which occurs. By means of these mechanisms the bidimensional aggregates are formed and cellular overlapping is not present. Since the behaviour of the mesoderm cells "in vitro" in some way could be comparable to their behaviour "in situ", the results here observed are discussed in relation to the conduct of mesoderm cells "in vivo".     

Molecular mechanisms of natural killer cell activation in response to cellular stress

Protection against cellular stress from various sources, such as nutritional, physical, pathogenic, or oncogenic, results in the induction of both intrinsic and extrinsic cellular protection mechanisms that collectively limit the damage these insults inflict on the host. The major extrinsic protection mechanism against cellular stress is the immune system. Indeed, it has been well described that cells that are stressed due to association with viral infection or early malignant transformation can be directly sensed by the immune system, particularly natural killer (NK) cells. Although the ability of NK cells to directly recognize and respond to stressed cells is well appreciated, the mechanisms and the breadth of cell-intrinsic responses that are intimately linked with their activation are only beginning to be uncovered. This review will provide a brief introduction to NK cells and the relevant receptors and ligands involved in direct responses to cellular stress. This will be followed by an in-depth discussion surrounding the various intrinsic responses to stress that can naturally engage NK cells, and how therapeutic agents may induce specific activation of NK cells and other innate immune cells by activating cellular responses to stress.     

Involvement of caveolin-1 in cholesterol enrichment of high density lipoprotein during its assembly by apolipoprotein and THP-1 cells

High density lipoprotein (HDL) is assembled by interaction of apolipoprotein A-I with human monocytic leukemia cell line THP-1 by removing cellular cholesterol and phospholipid. Although the HDL formed with undifferentiated THP-1 cells contained only phosphatidylcholine and almost no cholesterol, the cells differentiated with phorbol 12-myristate 13-acetate (PMA) generated HDL enriched in cholesterol. The extent of cholesterol enrichment related to the cellular cholesterol level in the differentiated cells, but only weakly in the undifferentiated cells. In contrast, the differentiation had no influence on the diffusion-mediated cellular cholesterol efflux. The undifferentiated cells expressed the messages of ATP-binding cassette transporter 1 and caveolin-1, at low levels, and the PMA-induced differentiation resulted in substantial expression of both messages. Caveolin-1 protein expression was also highly induced by the PMA treatment of THP-1 cells. When the cells were treated with the antisense DNA of caveolin-1 and differentiated, both caveolin-1 synthesis and cholesterol incorporation into the HDL were reduced in parallel to generate the cholesterol-poor HDL.We concluded that caveolin-1 is involved in enrichment with cholesterol of the HDL generated by the apolipoprotein-cell interaction. This function is independent of the assembly of HDL particles with cellular phospholipid and of nonspecific, diffusion-mediated efflux of cellular cholesterol.     

Novel spectral method for the study of viral carcinogenesis in vitro

Fourier transform infrared (FTIR) spectroscopy is a unique technique for the laboratory diagnosis of cellular variations based on the characteristic molecular vibrational spectra of the cells. Microscopic FTIR was used to investigate spectral differences between normal and malignant fibroblasts transformed by retrovirus infection. A detailed analysis showed significant differences between cancerous and normal cells. The contents of vital cellular metabolites were significantly lower in the transformed cells than in the normal cells. In an attempt to identify the cellular components responsible for the observed spectral differences between normal and cancerous cells, we found significant differences between DNA of normal and cancerous cells.     

Interrelation between cellular rRNA content and regulation of the cell cycle of normal and transformed mouse cell lines

The relation between cellular rRNA content, as a measure of cell size, and the regulation of the cell cycle has been investigated for swiss 3T3 and the spontaneously transformed swiss 3T6 cell line. It is shown that the characteristic of percent of quiescent cells stimulated into the cell cycle versus cellular rRNA content is basically different for 3T3 and 3T6 cells: 3T3 cells do not enter the cell cycle below a certain threshold of cellular rRNA content, whereas 3T6 cells start proliferation without a substantial increase of rRNA. These data are interpreted as consistent with transformation of 3T6 cells being in essence their uncoupling from the requirement of normal cells of passing over a threshold of cellular rRNA content (cell size) before initiating DNA-replication.     

How stem cells remember their past

Somatic stem cells are required for tissue development, homeostasis, and repair. Recent data suggested that previous biographical experiences of individual stem cells influence their behavior in the context of tissue formation and govern stem cell responses to external stimuli. Here we provide a concise review how a cell's biography, for example, previous rounds of cell divisions or the age-dependent accumulation of cellular damage, is remembered in stem cells and how previous experiences affect the segregation of cellular components, thus guiding cellular behavior in vertebrate stem cells. Further, we suggest future directions of research that may help to unravel the molecular underpinnings of how past experiences guide future cellular behavior.     

Apoptotic regulation of epithelial cellular extrusion

Cellular extrusion is a mechanism that removes dying cells from epithelial tissues to prevent compromising their barrier function. Extrusion occurs in all observed epithelia in vivo and can be modeled in vitro by inducing apoptosis in cultured epithelial monolayers. We established that actin and myosin form a ring that contracts in the surrounding cells that drives cellular extrusion. It is not clear, however, if all apoptotic pathways lead to extrusion and how apoptosis and extrusion are molecularly linked. Here, we find that both intrinsic and extrinsic apoptotic pathways activate cellular extrusion. The contraction force that drives cellular extrusion requires caspase activity. Further, necrosis does not trigger the cellular extrusion response, but instead necrotic cells are removed from epithelia by a passive, stochastic movement of epithelial cells.