Eukaryotic Cell Proliferation Research Articles

Is hypusine essential for eukaryotic cell proliferation?

Hypusine [N epsilon-(4-amino-2-hydroxybutyl)-L-lysine] is a most remarkable amino acid, occurring in all eukaryotic cells, yet occupying only a single position in one protein, eukaryotic protein synthesis initiation factor 5A (eIF-5A). The unusual structure of hypusine, its derivation from the polyamine spermidine, and its increased formation in response to growth stimulation, as well as its limited occurrence in the highly conserved amino acid sequence of eIF-5A, have aroused keen interest in the biological significance of its existence and in its relationship to eIF-5A function.

Carbon source induces growth of stationary phase yeast cells, independent of carbon source metabolism.

Nutrients regulate the proliferation of many eukaryotic cells: in the absence of sufficient nutrients vegetatively growing cells will enter stationary (G0 like) phase; in the presence of sufficient nutrients non-proliferative cells will begin growth. Previously we have shown that glucose is the critical nutrient which stimulates a variety of growth-related events in the yeast Saccharomyces cerevisiae (Granot and Snyder, 1991). This paper describes six new aspects of the induction of cell growth events by nutrients in S. cerevisiae. First, all carbon sources tested, both fermentable and non-fermentable, induce growth-related events in stationary phase cells, suggesting that the carbon source is the critical nutrient which stimulates growth. Second, the continuous presence of glucose is not necessary for the induction of growth events, but rather a short 'pulse' of glucose followed by an incubation period in water will induce growth events. Third, growth stimulation by glucose occurs in the absence of the SNF3 high affinity glucose transporter. Fourth, growth stimulation occurs independent of carbon source phosphorylation and carbon source metabolism. Fifth, growth induction by carbon source does not require protein synthesis or extracellular calcium. Sixth, following stimulation by carbon source, the cells remain induced for more than 2 h after removal of the carbon source. We suggest a general model in which different carbon sources act as signals to induce the earliest growth events during or following its entry into the cell and that these growth events do not depend upon metabolism of the carbon source.

An apparent growth factor modulation of marine dinoflagellate excystment

The effects of growth factors on Alexandrium tamarense (Halim) Baleen excystment were analysed under laboratory conditions. The addition of 10 ng·ml −1 of platelet-derivated growth factor (PDGF) or 10% of fetal bovine serum (FBS), which are potent mitogens used to increase eukaryotic cell proliferation in culture, caused a statistically significant increase in excystment. While in the unsupplemented medium scarcely any excystment took place during the first days, most of the excystment in the supplemented medium took place during the first days of treatment with growth factors.

Calmodulin and cell cycle control

Previous studies have indicated a role for the calcium receptor calmodulin in the control of eukaryotic cell proliferation. Using a molecular genetic approach in the filamentous fungus Aspergillus nidulans we have shown that CaM is required for cell cycle progression at multiple points in the cell cycle. Construction of an A nidulans strain conditional for calmodulin expression reveals that this protein is required during G1/S and for the initiation of mitosis. A lack of calmodulin results in cell cycle arrest, and a failure in polar growth that accompanies germination of A nidulans spores. In addition, increased expression of calmodulin in this organism permits growth at suboptimal calcium concentrations, indicating that cell growth is coordinately regulated by calcium and calmodulin. Together these results indicate that calmodulin-dependent processes may be conserved between A nidulans and vertebrate cells, and suggest that this approach may allow us to elucidate the molecular mechanism underlying calmodulin-regulated control of cell proliferation.

Fundamentals of Cell Proliferation: Control of the Cell Cycle

Cell proliferation in higher eukaryotes is controlled by the extracellular environment and the state of differentiation. Many cells exist in a nondividing growth state termed quiescence. Some quiescent cells cannot proliferate and are said to be terminally differentiated. Others can be stimulated to divide in response to environmental signals or when cell replacement is needed. Finally, some cells undergo continual proliferation and differentiation.Growth regulatory factors generally act at specific stages of the cell cycle, most commonly during the first gap phase of the cell cycle. Once cells initiate DNA synthesis, they are generally committed to complete DNA replication. After DNA synthesis, additional signals determine whether cells in the last gap phase proceed through mitosis.In recent years, genes that appear to be critical for progression through the first two gap phases have been identified. Many are proto-oncogenes and therefore can neoplastically transform certain cells when mutated or inappropriately expressed. Growth factors that stimulate proliferation induce the expression of several proto-oncogenes; growth inhibitory factors often suppress proto-oncogene expression.As cells differentiate, the response to extracellular factors changes. In many cases, this may be due to intracellular controls that alter the response of certain proto-oncogenes to external signals.

Surface Membrane-Associated Regulation of Cell Assembly, Differentiation, and Growth

The role of the surface membrane in regulating proliferation and differentiation of eukaryotic cells is highly complex. Proximal cell-cell interactions are involved, including physical contact via junctional complexes, exfoliation of surface membrane vesicles, proteolytic cleavage of membrane-bound precursors, and exocytosis of soluble proteins (see Fig 5). At another level, the surface expresses receptors and other proteins that mediate cell-cell adhesion, a multistep event that may be sufficient to activate some growth-associated genes and alter cell shape. The evidence indicates that together with secreted growth factors, surface membrane-associated molecules play a dynamic role in regulating multicellular assembly and cell differentiation and growth.

Surface membrane-associated regulation of cell assembly, differentiation, and growth

The role of the surface membrane in regulating proliferation and differentiation of eukaryotic cells is highly complex. Proximal cell- cell interactions are involved, including physical contact via junctional complexes, exfoliation of surface membrane vesicles, proteolytic cleavage of membrane-bound precursors, and exocytosis of soluble proteins (see Fig 5). At another level, the surface expresses receptors and other proteins that mediate cell-cell adhesion, a multistep event that may be sufficient to activate some growth- associated genes and alter cell shape. The evidence indicates that together with secreted growth factors, surface membrane-associated molecules play a dynamic role in regulating multicellular assembly and cell differentiation and growth.

Effects of In Vitro Electrical Stimulation on Enhancement and Suppression of Malignant Lymphoma Cell Proliferation

The suppression and enhancement of proliferation of mouse EL4 lymphoma cells varied significantly when the cells were electrically stimulated within a narrow range of low-level direct current. With the use of platinum electrodes, the suppression characteristics were observed over a narrow range, or window, of direct currents centered at approximately 17 microA. Enhancement characteristics were observed over a broad range of low-level direct currents (approximately 0.1 to 10 microA). These results indicate that the proliferation of certain eukaryotic cells may be directly controlled by stimulation with low-level direct current.

HP 0.35, a cephalosporin degradation product is a specific inhibitor of lentiviral RNAses H

Penicillins, cephalosporins and other betalactam antibiotics are widely used antibacterial drugs. Recently it was found that some of them also have effects on proliferating eukaryotic cells (Neftel, K.A. and Hübscher, U. (1987) Antimicrob. Agents Chemother. 31, 1657-1661), and one such effect was shown to be the inhibition of DNA polymerase alpha (Huynh Do,U., Neftel, K.A., Spadari, S. and Hübscher, U. (1987) Nucl. Acids Res. 15, 10495-10506). The data suggested that degradation products of betalactam antibiotics were responsible for the inhibitory effect on DNA polymerase alpha. There is some confirmation at the structural level, since we found that penicillin binding proteins, the natural target of the cephalosporins, share amino-acid homologies to DNA polymerases and also to reverse transcriptase from HIV1 (Hafkemeyer, P., Neftel, K.A. and Hübscher, U. Meth. Find. Exp. Clin. Pharmacol. 12, 43-46, 1990). We have purified and determined the structure of one product from the cephalosporin Ceftazidim and found one molecule (HP 0.35) that did not interfere with eukaryotic cell proliferation but rather had a specific inhibitory effect on the RNase H activity of human immunodeficiency virus 1 (HIV1) and feline immunodeficiency virus (FIV) reverse transcriptases, while the DNA polymerising activity of these enzymes was not affected. RNases H from HeLa cells, calf thymus and Escherichia coli on the other hand were much less affected by HP 0.35. The inhibitory concentration of 50% (IC50) was more than 10 times lower compared to those of all cellular RNases H. We therefore tested the effect of HP 0.35 on in vitro lentivirus infection as exemplified by FIV-infection of CD(4+)-cat lymphocytes in cell culture. Under conditions where cell proliferation was absolutely unaffected, HP 0.35 was able to inhibit FIV-infection in CD(4+)-cat lymphocytes. Moreover, preincubation of these lymphocytes with HP 0.35 rendered the cells completely unsusceptible to FIV-infection. These data suggest that a degradation product of a clinically used betalactam antibiotic might represent an effective inhibitor class for lentiviral RNase H.

A lymphocyte blastogenesis inhibitory factor (LBIF) reversibly arrests a human melanoma cell line, A375, at G 1 and G 2 phases of cell cycle

A lymphocyte blastogenesis inhibitory factor, LBIF, has been found in the culture supernatant of a human macrophage-like cell line, U937. The factor has been purified by fast protein liquid chromatography. Partial amino acid sequencing analysis showed that LBIF was a novel immunoregulatory factor. Recent study has demonstrated that LBIF possesses a remarkable tumor growth inhibitory activity. In this study, the cell growth inhibitory activity of LBIF was characterized on the proliferation of a human melanoma cell line A375 in vitro. LBIF strongly inhibits the proliferation of A375 cells. The inhibitory activity was cytostatic and reversible by Day 5 although the lethal effect became apparent at Day 7. Cell cycle analysis by flow cytometry showed that LBIF arrested A375 cells at both G 1 and G 2/M phases. Mitotic index analysis indicated that A375 cells were arrested in G 1 and G 2 phases. LBIF function was not attributed to the elevation of intracytoplasmic cyclic-AMP levels. Thus, these results suggest that LBIF plays an important role in controlling cell cycle and there is a similarity between the mechanisms of G 1 and G 2 arrests in eukaryotic cell proliferation. LBIF-induced reversible cell-cycle arrest of A375 cells can be a useful system to analyze the signal transduction for cell proliferation and cell-cycle arrest.

Epidermal Growth Factor Induction of Cellular Proliferation and Protooncogene Expression in Growth-Arrested Rat H4IIE Hepatoma Cells: Role of Cyclic Adenosine Monophosphate

The precise molecular events involved in growth factor-mediated cell proliferation in eukaryotes have not been entirely elucidated. Identification and characterization of the itnracellular molecular signaling systems linking growth factor function with nuclear events would provide insight into the regulatory mechanisms governing eukaryotic cell growth. In this report, we demonstrate that serum-deprived rat H4IIE hepatoma cells enter a quiescent state and remain viable in the absence of serum for up to 7 days. These cells can be stimulated to transverse the cell cycle and proliferate in response to epidermal growth factor (EGF) after a 24-h lag phase. We were able to completely mimic the mitogenic effects of EGF with 8-p-chlorophenylthio-cAMP (8-CPT-cAMP) but only partially with N6-(Bu)2-cAMP. EGF and 8-CPT-cAMP together induce a synergistic increase in H4IIE hepatoma cell proliferation. The calcium ionophore A23187 and the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate had little effect on H4IIE cell proliferation. EGF treatment led to a rapid and transient increase of intracellular cAMP concentration. Both 8-CPT-cAMP and EGF were also equally effective in causing a rapid and transient induction of c-fos and c-myc protooncogene mRNA levels when added to growth-arrested H4IIE cells while A23187, N-(Bu)2-cAMP, and 4 beta-phorbol 12-myristate 13-acetate were significantly less effective. Both EGF and 8-CPT-cAMP affect protooncogene expression in growth-arrested rat H4IIE hepatoma cells primarily at the transcriptional level. Localization and semi-quantification of nuclear pp55c-fos and 63 (kilodalton)-myc protooncoproteins by immunocolloidal gold electron microscopy revealed that EGF and/or 8-CPT-cAMP treatment of quiescent H4IIE hepatoma cells led to a marked and rapid nuclear accumulation of these proteins in discrete nuclear substructures. Cummulatively, these results suggest that cAMP participates in the intracellular signaling system mediating the mitogenic and protooncogene inducing effects of EGF on growth-arrested rat H4IIE hepatoma cells.

Specific induction of secreted proteins by transforming growth factor-beta and 12-O-tetradecanoylphorbol-13-acetate. Relationship with an inhibitor of plasminogen activator.

To examine the mechanisms by which transforming growth factors (TGFs) regulate the proliferation of eukaryotic cells, five cell lines, from different species and tissues, were treated with three agents that inhibit DNA synthesis and proliferation: BSC-1 cell-derived growth inhibitor (GI/TGF-beta), platelet-derived transforming growth factor-beta (TGF-beta), and 12-O-tetradecanoylphorbol-13-acetate. The cell lines tested were mink lung CCL 64 epithelial cells, Maloney sarcoma virus-transformed CCL 64.1, monkey kidney BSC-1 epithelial cells, human epidermoid A431 cells, and mouse embryo AKR-2B (clone 84A) cells. All cell lines responded to one or more of these agents by synthesizing and secreting a 48 to 51-kDa protein (IIP48). The TGF-beta s and 12-O-tetradecanoylphorbol-13-acetate had little or no effect on the incorporation of [35S] methionine into other secreted proteins or on the pattern of [35S]methionine-labeled intracellular proteins analyzed by one-dimensional, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maximum increase in induction of IIP48 varied from 2-fold to greater than 800-fold compared with the controls and occurred within 6 h of adding GI/TGF-beta to CCL 64 cells. Actinomycin D, alpha-amanitin, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole selectively decreased both the control and induced levels of IIP48 even after as little as 6 h of incubation. Thus, it appears that IIP48 mRNA turns over rapidly. Induction of IIP48 was dissociated from the inhibition of DNA synthesis by GI/TGF-beta. However, we found that epidermal growth factor and GI/TGF-beta act synergistically to increase the secreted level of IIP48. Others have shown that epidermal growth factor and TGF-beta act synergistically to stimulate growth of cells in agar. IIP48 from CCL 64, BSC-1, and AKR-2B cells is specifically immunoprecipitated by antibody to bovine plasminogen activator inhibitor. We found previously that TGF-beta also inhibits the production of major excreted protein, a thiol protease. It is proposed that TGF-beta is able to promote anchorage-independent growth of untransformed cells because of its ability to inhibit the production of secreted proteases and to increase the production of protease inhibitors.

Control of DNA replication and cell proliferation in eukaryotes by aphidicolin.

The mycotoxin aphidicolin specifically inhibits nuclear DNA synthesis in eukaryotic cells by intereacting with the replicative DNA polymerase alpha. The drug does not bind directly to DNA nor does it interfere with RNA, protein and nucleic acid precursor synthesis. Aphidicolin offers a new tool for analyzing the mechanism of DNA replication and repair in eukaryotes and for studying the role of eukaryotic DNA polymerases. The drug might also be valuable therapeutically for controlling excessive cell proliferation without adverse effects upon non-multiplying cells. We describe here how to synchronize DNA synthesis in human cells by a double block with aphidicolin and the properties of two human cell lines resistant to the drug.

Demonstration of a proliferation regulatory factor (PRF) in normal human urine, and its purification.

A material(s) affecting the proliferation of eukaryotic cells, such as C3H2K cells, and prokaryotic cells, such as E. coli, was found in the urine of normal healthy men and was concentrated in a certain fraction, "lysin-Sepharose eluate," of the urine. The material was named proliferation regulatory factor, PRF. With increase in the concentration of PRF, the proliferation rate of normal eukaryotes and prokaryotes increased to maxima of no more than twice those of untreated cells. On further increase in the concentration of PRF the growth rates of these cells decreased to the rates of untreated cells. PRF enhanced the rates of synthesis of DNA and RNA in C3H2K cells about 2-fold, but strongly suppressed the rates of growth, DNA synthesis, and RNA synthesis of cancerous cells, such as HeLa and MH134/M cells. PRF was purified from the "lysine-Sepharose eluate" by DEAE-cellulose column chromatography and gel filtration on a Sephadex G-100 column. PRF consisted of three molecules with molecular weights of 41,000, 7,800, and 5,100, which were named PRF-I, II, and III, respectively. The lysine-Sepharose eluate and purified PRF's had similar effects on the proliferation of eukaryotes and prokaryotes.

Cations and calmodulin in normal and neoplastic cell growth regulation.

The purpose of this presentation is to review pertinent literature pertaining to the role of divalent cations and calmodulin in regulating growth of nonneoplastic and neoplastic cells and to examine the anticancer efficacy of some calmodulin inhibitors. Although normal eukaryotic cell replication and proliferation is closely controlled by a complex system of endogenous substances, it is likely that the coordination of purposeful interactions between these substances is the ultimate responsibility of two groups of cellular components, namely the divalent cations Ca2+ and Mg2+ and the versatile intracellular Ca2+-binding protein calmodulin (CaM). When free Ca2+ enters normal cells, it acts as a positive signal for proliferation; this action appears to be specifically associated with the late G1 phase, just prior to DNA synthesis. This period is designated G1/S and is considered to contain Pardee's "restriction point." Reduction of extracellular Ca2+ concentrations between physiological levels (1-0.1 mM) results in gradually reduced rates of cell proliferation; at Ca2+ concentrations of 0.1 mM or less, normal cell proliferation is reversibly inhibited. Since an extracellular concentration of about 0.7 mM Mg2+ is required for Ca2+ to initiate cell replication, it has been proposed that Ca2+ and Mg2+ act in concert via a common mechanism, however, in contrast to Ca2+, Mg2+ appears to be required throughout the entire cell cycle. Intracellular Ca2+ can activate CaM which, in turn, can modulate various cellular processes that affect cell proliferation, including cyclic nucleotide metabolism, protein phosphorylation, polyamine metabolism, prostaglandin metabolism, Ca2+ transport, DNA synthesis, and microtubular function including mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)

P1,P4-Di(adenosine-5')tetraphosphate inhibits phosphorylation of immunoglobulin G by Rous sarcoma virus pp60src.

Di(adenosine-5')oligophosphate nucleotides of general structure ApnA (n = 2-6) inhibited phosphorylation of immunoglobulin G from tumor-bearing rabbits (TBR IgG) by pp60src protein kinase purified from Rous sarcoma virus-transformed rat tumor cells. Ap4A, a nucleotide associated with eukaryotic cell proliferation, was one of the most effective inhibitors in the series, causing 50% inhibition of TBR IgG phosphorylation at 15 microM. Ap4A inhibited pp60src-dependent phosphorylation of TBR IgG in solution and immunoprecipitates, as well as the phosphorylation of tubulin, microtubule-associated proteins, and vinculin. Under similar assay conditions, Ap4A did not inhibit phosphorylation of histone H2b by cAMP- or cGMP-dependent protein kinases. Ap4A appears to interact noncovalently with the enzyme, because removal of pp60src by immunoprecipitation from solutions containing Ap4A restored activity to uninhibited levels. A 100-fold increase in ATP (4-400 nM) caused a 13-fold increase in the 50% inhibitory concentration of Ap4A (2.5-33 microM), consistent with the interpretation that Ap4A competes for an ATP-binding site on the pp60src molecule. The simplest explanation of these results is that Ap4A binds to the phosphodonor site for ATP.

Spermidine requirement for cell proliferation in eukaryotic cells: structural specificity and quantitation.

Six structural homologs of spermidine and five of its precursor, putrescine, were studied for their ability to prevent cytostasis of cultured L1210 leukemia cells induced by alpha-difluoromethylornithine (DFMO), a specific inhibitor of putrescine biosynthesis. High-performance liquid chromatography and competition studies with spermidine indicated that the homologs, which vary in the length of the carbon chain separating the amines, penetrated the cells. The structural specificity of the spermidine carrier was defined. Three of the six spermidine homologs supported cell growth during a 48-hour incubation in the presence of DFMO, indicating that a two-carbon extension of spermidine structure was tolerated for biological function. Two of the five putrescine homologs supported growth after being converted by the cells to their respective spermidine homologs. The central nitrogen of spermidine appears to be essential for function since diamines of chain length comparable to that of spermidine did not prevent DFMO cytostasis. No more than 15 percent of the spermidine normally present in L1210 cells was required for cell proliferation in the presence of DFMO.

Susceptibility of normal and transformed cell lines to cytostatic and cytocidal effects exerted by macrophages.

Activated, nonimmune macrophages exerted profound effects on the proliferation and viability of eukaryotic target cells in vitro. Pronounced macrophage-mediated cytostasis was exerted on every rapidly proliferating cell line examined, irrespective of transformation, species derivation, cell type, or growth characteristics. However, the magnitude of cytostasis effected differed markedly among the 40 cell lines tested. There was no evident correlation between susceptibility to cytostasis and degree of transformation. Transformed cell lines with high and with low malignant attributes were affected equally. A comparable pattern was discerned for cytocidal effects of macrophages, in which the susceptibility of transformed targets was independent of the degree of malignancy.

Evidence that mitogenic lectins induce changes in lymphocyte membrane fluidity

MITOGENIC plant lectins, such as phytohaemagglutinin (PHA) and concanavalin A (con A), are useful tools to study the control of cell proliferation in lymphocytes. Recent evidence suggests that these mitogenic lectins trigger mitogenesis at the cell membrane1,2. It is likely that an understanding of the mechanisms by which these lectins stimulate lymphocytes will provide important clues to more general problems of the regulation of proliferation in eukaryotic cells.

Deoxyribonucleic acid synthesis, mitosis, and skeletal muscle differentiation

In the past, it was generally thought that processes of differentiation and proliferation in eukaryotic cells were antagonistic, the implication being that somehow the two processes cannot go on simultaneously within the same cell. There are, however, many examples of synthesis of deoxyribonucleic acid and of specialized cell proteins occurring simultaneously in the same cell (1-5). It has become clear in recent years that it would be more accurate to say that there are several differentiating systems in which special relationships exist between the events leading to overt differentiation and cell proliferation. Some of the better studied examples are in erythroid differentiation, where a programmed sequence of cell division occurs (6-9); in formation of tubular gland cells, in which division is required for the formation and subsequent synthesis of ovalbumin in the chick oviduct (10, 11); in viral transformation of cells, where cell division is required to establish this state (12-15); in the differentiation of mammary gland epithelium, where cell division must occur in the presence of hydrocortisone in order for prolactin to stimulate milk protein synthesis (16, 17); and in the differentiation of skeletal muscle cells, where cells synthesizing contractile proteins do not divide (18, 19). For several years we have been exploring the relationship of cell proliferation and differentiation in skeletal muscle cells differentiating in vitro. We describe this relationship and relate some of our general conclusions about deoxyribonucleic acid synthesis in differentiating myogenic cells. DISCUSSION