Initiation Of Cell Proliferation Research Articles

Influence of p-dimethylaminoazobenzene and doxorubicin on tobacco cell growth (Nicotiana tabacum L.)

Callus growth, in tobacco pith tissue culture, is activated by p-dimethylaminoazobenzene (p-DAAB), whose carcinogenic properties are well known. Ultrastructural changes, appearing in a period prior to initiation of cell proliferation, occur earlier and more intensely in the presence of the carcinogen. This also influences changes in non-histone chromatin proteins (NHCP). Doxorubicin (DR), an anti-tumor drug, inhibits callus growth and modifies the pattern of NHCP.

Nucleotide pools in suspension-cultured cells of Datura innoxia

The concentrations of 17 nucleotides and three nucleosides have been determined in a batch suspension culture of Datura innoxia using a new procedure for extraction, purification and high-performance liquid chromatography separation of these compounds. The nucleotide pools change appreciably in the different phases of growth. These changes indicate the preparation for and initiation of cell proliferation, and reflect metabolic events during cell division, cell elongation and starvation. The main components of the nucleotide pool are uracil nucleotides, with uridine 5'-diphosphate sugars as the predominant fraction, and the adenine nucleotides. Although their concentrations vary by a factor of more than 6 the ratio of the uracil to adenine nucleotides is kept fairly constant during growth. The energy charge is maintained at a rather high value. The correlation of these events with nutrient uptake and macromolecular synthesis by the batch culture is presented in the following paper.

Mitogens trigger a calcium-independent signal for proliferation in phorbol-ester-treated lymphocytes

The activation of T lymphocytes by mitogens requires at least two signals; the first, delivered to T cells by a mitogen in conjunction with accessory cells (monocytes/macrophages), leads to the generation of the second signal, interleukin-2 (IL-2). The first signal also induces the expression of IL-2 receptors on the surface of a subpopulation of T cells; binding of IL-2 to its receptor then initiates a cascade of events culminating in DNA synthesis by these cells. Certain compounds act synergistically with mitogens in promoting T-cell proliferation by substituting for the activities of interacting cells or their products. For example, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) has been shown to restore the ability of macrophage-depleted T-cell populations to respond to mitogenic lectins. Transmembrane fluxes of calcium, leading to increased free cytosolic calcium concentrations ([Ca2+]), have been demonstrated following mitogen binding to lymphocytes and have been implicated in the initiation of cell proliferation. We show here that the effect of TPA on lymphocyte proliferation occurs in the absence of extracellular Ca2+ or detectable changes in [Ca2+]i, but only in the presence of mitogens. This suggests that in cells which have been incubated with the phorbol ester, mitogens can induce proliferation by a calcium-independent signal.

Oncogenes, ions, and phospholipids.

Recent discoveries in tumor virology, lipid biochemistry, and ion transport studies promise to revolutionize our understanding of cell proliferation, differentiation, and tumorigenesis. A model is proposed, based on similar schemes presented recently by others, that incorporates these discoveries and provides a focus for future research on the functions of oncogene proteins. The model suggests that the early (competence) events in the initiation of cell proliferation are triggered by activation of phosphatidylinositol (PI) turnover, which releases two second messengers, 1,2-diacylglycerol (1,2-DG) and inositol-1,4,5-trisphosphate (IP3). PI turnover is proposed to be regulated by the oncogene protein kinases (src, ros, abl, fps) either directly (acting as PI kinases) or indirectly (as tyrosine kinases). The IP3 triggers Ca2+ release from internal stores, and the elevation of cytosolic Ca2+ acts synergistically with 1,2-DG to activate the Ca2+- and phospholipid-dependent kinase C. Kinase C copurifies with the receptor for the tumor-promoting phorbol esters. It is suggested that kinase C then activates the Na+-H+ exchange system, resulting in an elevation of cytosolic pH and Na+, and that these ionic signals (including the change in Ca2+), either in concert or individually, induce further events, including expression of the protooncogene c-myc, which together commit the cell to initiate replication. Evidences in support of this model are reviewed, together with complications indicating its present inadequacies, particularly recent data suggesting that 1,2-DG may activate tyrosine kinases independent of kinase C.

14C-metabolism during callus induction in tobacco

Tobacco pith-phloem explants and callus were incubated in 14C-glucose, 14C-acetate or 14C-bicarbonate on different days in culture in the dark. 14CO2 production and 14C incorporation into ethanol-insoluble components were generally greater in the subcultured callus than in the pith-phloem explants during days 0 to 5 in culture. Greatest radioactivity from all substrates was in the ethanol-soluble portion, which was further fractionated into lipids, amino acids, sugars and organic acids. Although incorporation into the different fractions varied with the substrate, the patterns of labelling were relatively similar in the two tissues. The greater wound metabolism in the subcultured callus in comparison to the pith-phloem explant during the induction phase of callus formation was correlated with the earlier visible initiation of cell proliferation in the subcultured tissue.

Increased cyclic GMP content in confluent and serum-restricted heart cell cultures exposed to polyamines.

Cyclic AMP (cAMP) and cyclic GMP (cGMP) have been implicated as intracellular signals in the transition from a resting to a growing state. This suggestion comes from observations showing that the addition of growth promoting factors to quiescent cell cultures causes a rapid and transient decrease in cAMP and an increase in cGMP contents [9, 11] and that exogenous cAMP or cGMP congeners reduce or stimulate cell growth respectively [6, 13]. In view of this antagonistic effect elicited by the two nucleotides, it has been suggested that a fall in cAMP/cGMP ratio might be the triggering event for the initiation of cell proliferation [6]. Since polyamines correlate positively with active cell division [7], a possible involvement of these biogenic polycations in the regulation of cellular cyclic nucleotide contents is worthwhile investigating. Our previous reports have shown indeed that in different cultured cell types, spermine, spermidine and putrescine, at relatively low doses, are able to reduce cAMP content [3] by increasing cAMP-dependent phosphodiesterase activity (cAMP-PDE) [4] and to counteract the action of different cAMP-mediated effectors [3]. Besides endogenous polyamines seem to be involved in the cAMP-mediated induction of cAMP-PDE, as observed in heart cell cultures [4]. This report shows that the addition of each individual polyamine to confluent and serum-restricted heart cell cultures, while lowering cAMP content, induces an early and rapid increase of cGMP content by reducing the rate of its degradation.

Evidence that microtubule depolymerization early in the cell cycle is sufficient to initiate DNA synthesis

Microtubule disrupting drugs initiated DNA synthesis in serum-free cultures of nonproliferating fibroblast-like cells. The addition of colchicine to chick, mouse and human fibroblasts in serum-free medium stimulated thymidine incorporation at least twofold, with a half-maximal concentration of 1 × 10 −7 M. This stimulation represented up to 75% of the maximal stimulation by thrombin and was paralleled by an increase in the percentage of labeled nuclei. Other microtubule disrupting drugs showed similar stimulation, whereas lumicolchicine had no effect. Indirect immunofluorescent staining of tubulin showed a correlation between microtubule depolymerization and initiation of DNA synthesis by these drugs. A 2 hr treatment with 10 −6 M colchicine caused complete disruption of the microtubular network and stimulated thymidine incorporation (measured 28 hr later) to an even greater extent than continuous colchicine exposure. A similar 2 hr exposure to 10 −6 M colcemid also stimulated thymidine incorporation and led to a 50% increase in cell number. Taxol, a drug which stabilizes cytoplasmic microtubules, blocks initiation of DNA synthesis by colchicine, indicating that microtubule depolymerization is necessary for this initiation. To determine if microtubule depolymerization is involved in stimulation of DNA synthesis by other growth factors, highly purified human thrombin was added to cells with or without colchicine. In no case did colchicine plus thrombin increase DNA synthesis above that of the maximal stimulation by thrombin alone. Furthermore, pretreatment of cultures with taxol (5 μg/ml) inhibited approximately 30% of the stimulation of thymidine incorporation by thrombin. Together, these studies demonstrate that microtubule depolymerization is sufficient to initiate both DNA synthesis and events leading to cell division and suggest that microtubule depolymerization may be a required step in initiation of cell proliferation by growth factors such as highly purified human thrombin.

A rationale for the loss of growth control during experimental bladder carcinogenesis

A rationale is presented which provides an explanation for the loss of growth control which is associated with the early phase of experimentally-induced bladder cancer. Two early events which occur in the urothelium following exposure to carcinogen, the focal loss of alkaline phosphatase activity and the initiation of cell proliferation, are both proposed to be the result of a defect in the interaction between glucocorticoid hormone and urothelium. The possible causes for this defect are discussed in terms of a defect in, or an interference with, the glucocorticoid-receptor mechanism.

An examination of the roles of cyclic nucleotides in the initiation of cell proliferation

The initiation of proliferative activity in proliferatively quiescent confluent monolayers of BALB/3T3 cell cultures by a fresh medium-serum replacement is independent of early (within minutes) fluctuations in total cellular cyclic AMP or cyclic GMP levels. However, the possibility of a later prereplicative cyclic AMP surge being involved in the initiation of DNA synthesis will be discussed.

Changes in Transcription of Endogenous Type-C Virus Genome During Mouse Liver Regeneration

In the present study the extent of endogenous, type-C virus genome transcription in normal and regenerating mouse liver was analysed by using the technique of nucleic acid hybridization. The RNA preparations from regenerating liver tissues collected at various intervals following partial hepatectomy, and from normal liver samples of BALB/c mice, were hybridized to 3H-DNA complementary to 60 to 70S RNA of an endogenous, N-tropic virus, released spontaneously from BALB/c mouse cells in culture. Although partial transcription of the endogenous virus genome can be clearly detected in normal liver, a significant increase in the level of virus-specific RNA synthesis in the regenerating liver, in comparison to normal liver, is apparent, following partial hepatectomy. This increase in virus-specific RNA synthesis attains its highest level just before the level of DNA synthesis in the regenerating liver reaches its maximum. These observations may indicate a qualitative or quantitative change in the endogenous type-C virus genome transcription pattern in hepatocytes, in response to partial hepatectomy and suggest that this change in the transcription pattern and the initiation of cell proliferation, in regenerating livers, are probably sequential and related events.

The Involvement of Cyclic Nucleotide Metabolism in the Initiation of Lymphocyte Proliferation Induced by Mitogens

Murine lymphocytes incubated with specific mitogens were examined for alterations in levels of intracellular cyclic AMP and cyclic GMP. Mitogenic concentrations of LPS elevate cyclic GMP levels; however, DxSO4, Poly I:C, PPD, Con A, and PHA show little immediate effect on cyclic GMP levels. Higher concentrations of all of these mitogens consistently elevate cyclic AMP levels. Exogenous cyclic GMP stimulates DNA synthesis in homozygous nude (nu/nu) spleen cultures but not in murine thymocyte cultures. Both LPS and cyclic GMP enhance the mitogenic response of thymocytes in the presence of suboptimal concentrations of Con A. Exogenous cyclic AMP inhibits mitogenic responses to both LPS and Con A; however, these inhibitory effects are partially reversed when cyclic GMP is also added to cultures. The mitogenic response of nu/nu spleen cells to LPS can be inhibited by high concentrations of Con A. This inhibition by Con A is also partially reversed by cyclic GMP, indicating that Con A may exert its effect by elevating intracellular levels of cyclic AMP. Thus, while lymphocytee mitogens may affect intracellular levels of both cyclic AMP and cyclic GMP, three effects show a consistent pattern. 1) Exogenous cyclic GMP is either mitogenic for B cells or agents that elevate cyclic GMP levels enhance the mitogenic response of T cells to Con A. 2) Increasing cyclic AMP levels inhibit cell proliferation induced by both B and T cell mitogens. 3) The inhibitory effects of cyclic AMP or cyclic AMP-elevating agents on mitogen-induced proliferation can be partially reversed by the presence of cyclic GMP. The opposing effects of these two cyclic nucleotides may reflect either their involvement in the initiation of cell proliferation or thei modulating effects on cell cycle events after the initiation process has been completed.

Serum can initiate DNA synthesis in cells rendered unresponsive to insulin and somatomedin.

AN understanding of growth control in animal cells requires an appreciation not only of cellular events important to multiplication, but also of exogenous agents which either initiate or influence the ‘decisions’ of a cell to reproduce. Serum has been reported to contain factors required for cell movement1, viability2, ion transport3 and control of cell density4. In addition, peptides from tissue or plasma have been identified (nerve growth factor (NGF)5, somatomedin6, non-suppressible insulin-like activity (NSILA)7, multiplication-stimulating activity (MSA)8, epidermal growth factor9, fibroblast growth factor10) which seem to have a physiological role in the initiation of cell proliferation. Somatomedin, NSILA and MSA are probably similar, if not identical, substances and, like NGF, seem to be structurally related to insulin11–13. The ability of insulin to initiate DNA synthesis in various cells14–17 may be due to cross reactivity of the hormone with receptor sites concerned with cell multiplication12. Recent reports suggest that serum contains additional substances that can influence the ability of a cell to respond to agents which initiate DNA synthesis10,18,19. While investigating the regulatory roles of insulin and serum in the initiation of DNA synthesis in quiescent chick embryo fibroblasts (CEF), we have found a distinctive difference in the initiation capacity of these agents after the inhibition of protein synthesis. We present here evidence for the existence of a regulatory component(s) in serum which recruits cells for growth from a stage in the cell cycle position (G0?) which is refractory to mitogenic peptides.

Initiation of cell proliferation in cultured mouse fibroblasts by prostaglandin F2alpha.

Prostaglandin (PG) F2alpha added to quiescent Swiss mouse 3T3 cell cultures initiates DNA synthesis and cell proliferation in a small proportion of the cells. Insulin, which markedly potentiates the effect of PGF2alpha on the initiation of cell division when added with PGF2alpha, causes a simultaneous reduction in intracellular cyclic AMP and an increase in cyclic GMP concentrations similar to those observed after serum addition. Prostaglandins E1 and E2 are less effective than PGF2alpha in initiating DNA synthesis and this effect is only observed at higher concentrations. Therefore, some prostaglandins can act as extracellular factors to regulate cell proliferation.

A micromethod for lymphoblastic transformation of mouse lymphocytes from peripheral blood.

Although several micromethods for culturing human peripheral lymphocytes exist (1–3), attempts to develop similar procedures for small laboratory animals have not been equally successful (4–6). First, mouse lymphocytes appear less responsive to PHA stimulation than human lymphocytes under their respective culture conditions (7, 8). Second, because cell suspensions from whole organs have generally been used from mice, direct comparisons to human peripheral lymphocytes are difficult; also, the mice had to be killed, thus eliminating them from further use or breeding experiments. Third, the culture systems for mouse lymphocytes employed varying amounts of heterologous serum (5, 6) or lacked appropriate standardization with respect to optimal culturing conditions and culture time. The lack of a fast and reproducible micromethod for lympho-blastogenic transformation in mice is regrettable because it precludes studies on the genetic control of transformation and initiation of cell proliferation without sacrific...

In Vitro Cultivation of Cells of the Oyster, Crassostrea virginica

Cardiac tissue cells from oysters (Crassostrea virginica), cultured in a salt-enriched medium, apparently required a higher salt concentration for initiation of cell proliferation than that used for mammalian tissue cultures. The presence of human serum and bovine amniotic fluid enhanced the cell proliferation. Evidence of amitosis was observed in the cultured cells.