bluuebunny/crossref_metadata_2025 huggingface dataset:
Clonal generation of multipotent and unipotent hemopoietic blast cell colonies in vitro
Authors: Keller Gordon , Holmes Wendy , Phillips Robert A.
Published: February 2005

Colonies with a unique compact morphology and displaying high replating potential develop in methycellulose cultures when bone marrow cells from hydroxyurea‐ or 5‐fluorouracil‐treated mice are cultured in the presence of syngeneic thymocytes. These compact colonies are clonal, originating from a single unit, the compact‐colony‐forming unit (CFU). Based on replating analysis, at least three groups of compact colonies exist: (1) those that do not replate, (2) those that replate and give only GM colonies, and (3) those that give rise to granulocyte‐macrophage (GM), erythroid (E), and mixed‐erythroid (MIX/E) colonies. Colonies in the last group have a mean size of 2,060 cells and generate an average of 244 colonies in secondary cultures. Velocity sedimentation studies showed the compact‐CFU to be similar in size to the MIX/E‐CFU, sedimenting at rates between 3.5 and 7 mm/hour. In addition to giving rise to large numbers of in vitro CFU, compact‐CFU are also able to generate day 8 spleen colony‐forming units (CFU‐S).
Partial characterization of the mitogenic action of pp60v‐src, the oncogenic protein product of the src gene of avian sarcoma virus
Authors: Durkin Jon P. , Whitfield James F.
Published: February 2005

NRK cells infected with a temperature‐sensitive, transformation‐defective mutant of avian sarcoma virus (ASV), tsLA23, are transformed at 36°C, but at 40°C they behave as nontransformed cells because of the inactivation of the abnormally thermolabile pp60v‐src product of the virus' transforming src gene. At 40°C, these tsLA23‐NRK cells were arrested in G1/G0 by severe serum deprivation. They were induced to enter G1, initiate DNA synthesis 7 or 10 hours later, and then divide as (1) nontransformed cells by adding serum or platelet‐derived growth factor (PDGF) at 40°C, or (2) transformed cells by lowering the temperature to a pp60v‐src‐activating 36°C without adding exogenous growth factor(s). The level of pp60v‐src kinase activity rose dramatically in these serum‐deprived cells within 30 minutes of lowering the temperature to the permissive 36°C, and it fell just as rapidly when the cells were returned to the restrictive 40°C. As little as a 2‐hour exposure to 36°C, with an attendant 2‐hour burst of pp60v‐src kinase activity, was enough to stimulate serum‐deprived tsLA23‐NRK cells to transit G1 and initiate DNA replication, but not to divide. Much more prolonged pp60v‐src activity was needed for these serum‐deprived cells to complete their cycle and divide. The prereplicative development of quiescent tsLA23‐NRK cells stimulated by serum or PDGF was accompanied by greatly increased protein synthesis and slightly decreased protein degradation, but the pp60v‐src‐stimulated cells progressed through G1 and initiated DNA replication without appreciably affecting the protein synthetic machinery of the cell. The cells stimulated by the mitogenic action of pp60v‐src, like the cells stimulated by serum, needed to activate early prereplicative genes in order to initiate DNA replication. The needed RNA transcripts induced by serum and pp60v‐src were produced with comparable efficiency, although it took longer for pp60v‐src‐stimulated cells to translate these transcripts and to initiate DNA replication, probably because of their unstimulated protein‐synthetic machinery.
Induction of cytochrome P‐450 isozymes in rat hepatoma‐derived cell cultures
Authors: Frey Alan B. , Rosenfeld Melvin G. , Dolan William J. , Adesnik Milton , Kreibich Gert
Published: February 2005

We have investigated the responsiveness of established rat hepatocyte cell cultures to inducers of cytochrome P‐450. One Reuber hepatoma‐derived line (Fu5‐C8), which under normal culture conditions produces no detectable cytochrome P‐450(MC) or cytochrome P‐450(PB)—the major cytochrome P‐450 isozymes induced by 3‐methylcholanthrene and phenobarbital, respectively‐ was tested for the ability to accumulate either cytochrome P‐450 isozyme in response to treatment with various xenobiotics. By immune‐precipitation from [35S]‐methionine‐labeled cell extracts, using monospecific anticyto‐chrome P‐450(MC) antybody or monoclonal anticytochrome P‐450(PB) antibody, it was demonstrated that these cells possess the capability to synthesize cytochrome P‐450(MC) in response to 3‐methylcholanthrene treatment, while none of the drug treatments caused the synthesis of detectable quantities of cytochrome P‐450(PB). RNA extracted from Fu5‐C8 cells directed the in vitro synthesis of immune‐precipitable cytochrome P‐450(MC) only after treatment of the cells with 3‐methylcholanthrene Kinetic analysis of the response of these cells to 3‐methylcholanthrene induction revealed detectable levels of immune‐precipitable cytochrome P‐450(MC) 2 h after drug treatment with maximal induction occurring between 12 and 16 h of exposure. Another cell line (HF 1.5), obtained originally by hybridization of Fao X H5 variants of a Reuber H35 hepatoma, produces cytochrome P‐450(MC) and also cytochrome P‐450(PB) constitutively, as determined by specific immune‐precipitation from labeled cell extracts. Exposure of confluent monolayers to either phenobarbital or 3‐methylcholanthrene resulted in an induction of cytochrome P‐450(PB) or cytochrome F‐450(MC), respectively. Double‐labeling immunofluorescence studies indicate that all cells in the culture produce albumin and most of the cells produce cytochrome P‐450(MC), but only a subset of cells synthesize cytochrome P‐450(PB). Our results demonstrate that some continuously dividing hepatocyte cell cultures retain the capacity to respond to xenobiotics, including phenobarbital, a response which is typically exhibited by fully differentiated liver cells. Such established hepatocyte cell cultures should prove useful for investigating the mechanism of induction of cytochrome P‐450(PB).
Retinoic acid alters subcellular compartmentalization of ATP pools in 3T3 cells but not in HeLa cells
Authors: Schroder Edward W. , Rapaport Eliezer
Published: February 2005

Retinoic acid (RA;β‐all‐trans) inhibits the proliferation of both murine 3T3 cells and human HeLa cells. Flow cytometric analyses of exponentially growing cultures show that 3T3 cells are inhibited during the S phase of their cell cycle, while HeLa cells show only a small increase in G1 phase cells. RA (10 μM) causes a 50% increase in total cellular adenosine triphosphate (ATP) pools of 3T3 cells, but not of HeLa cells. We have previously demonstrated that the effects of RA on cellular ATP pools of 3T3 cells are directly related to its inhibition of cellular growth, and now report data which provide a biochemical basis for this process. Established procedures were utilized to investigate the effects of RA on the functional compartmentalization of the nuclear ATP pool which serves as a precursor for RNA synthesis in these cells, and which is shown to be a small pool in comparison with cytoplasmic ATP pools. Expansion of total cellular ATP pools by 1 mM of exogenously supplied unlabeled adenosine is ineffective in reducing the subsequent incorporation of [3H]adenosine into RNA of 3T3 cells. Similar treatment of HeLa cells yields a modest reduction in the incorporation of [3H]adenosine into RNA. RA treatment of HeLa cells does not affect the preferential uptake of exogenous [3H] adenosine into the immediate precursor ATP pool for RNA synthesis. RA treatment of 3T3 cells markedly reduces the incorporation of [3H] adenosine into RNA, indicating a lesser degree of functional compartmentalization of the nuclear ATP pool. Similar conclusions are drawn from correlations of the specific radioactivities of total cellular [3H] ATP pools and the levels of incorporation of radioactive label into cellular RNA. In addition, pulse‐chase experiments show that RA‐treated 3T3 cells continue to incorporate radioactive label from pools prelabeled with [3H]adenosine despite the presence of a large excess of unlabeled adenosine in the chase medium. Control 3T3 and both control and RA‐treated HeLa cells cease to incorporate label immediately upon the start of the chase, suggesting that the functional precursor ATP pool for RNA synthesis is small and readily diluted. These data suggest that RA decreases the degree of functional compartmentalization for 3T3, but not HeLa cell ATP pools, and provides a probable mechanism for expansion of nuclear ATP pools of 3T3 cells. The expanded nuclear ATP pools may provide the biochemical mechanism for the inhibition of DNA synthesis during the S phase of the 3T3 cell cycle.
Changes in nuclear antigens during reactivation of chick erythrocyte nuclei in heterokaryons
Authors: Nyman Ulf , Lanfranchi Gerolamo , Bergman Mathias , Ringertz Nils R.
Published: February 2005

Reactivation of dormant chick erythrocyte (CE) nuclei was studied by fusing chick red cells with rat myoblasts, HeLa cells, and chick fibroblasts. Heterokaryons representing different stages of nuclear reactivation were fixed and examined for nuclear antigens using polyclonal patient autoantisera reacting with mammalian (human, mouse, and rat) nuclear envelope, nucleoplasm, and chromatin (DNA) antigens. Reactivation of CE nuclei was associated with marked changes in nuclear antigenicity. In rat myoblast and HeLa heterokaryons the CE nuclei acquired mammalian nucleoplasmic and nuclear envelope antigens in the corresponding nuclear subcompartments. Drastic changes in nuclear antigenicity were noted also in heterokaryons stained with DNA antisera. The compact chromatin of nuclei in mature chick erythrocytes showed little binding of DNA antibodies. Isolated nuclei on the other hand gave a strong immunofluorescence. CE nuclei in heterokaryons were strongly positive during the early stages of nuclear reactivation but then exhibited decreased reactivity. An unexpected finding was a marked reduction in the capacity of mammalian nuclei in heterokaryons to bind DNA‐antibodies. This observation is discussed in relation to the previous finding that in CE‐heterokaryons these nuclei often show reduced transcription and replication. The present results indicate that in heterokaryons both types of nuclei exchange macromolecules of regulatory importance via the common cytoplasm.
Regulation of thrombospondin secretion by cells in culture
Authors: Mumby Susanne M. , Abbott‐Brown Debbie , Raugi Gregory J. , Bornstein Paul
Published: February 2005

Thrombospondin (TS), a 450,000 molecular weight glycoprotein, is released from α‐granules of thrombin‐activated platelets and is secreted and incorporated into the extracellular matrix by several cell types in culture. We have examined the effects of cell density and transformation on the production of TS in cell culture. The levels of TS, per cell, in the culture medium of endothelial cells, smooth muscle cells, and fibroblasts were greater at lower cell densities; in fibroblasts the levels of two other extracellular matrix proteins, fibronectin and collagen, were unaffected by cell density. Our evidence indicates that the higher levels of TS in the culture medium, determined for lower‐density cells, were achieved by an increased secretion of the protein rather than by a reduction in degradation or incorporation into the extracellular matrix. TS production by normal and transformed Wl‐38 fibroblasts was the same, although the fibronectin level in the culture medium of the transformed cells was substantially decreased. These findings suggest that the production of TS by cells in culture is regulated in a different fashion from that of fibronectin or collagen.
Effects of sodium butyrate on growth and cell‐cycle kinetics of cultured rabbit articular chondrocytes
Authors: Larno Suzanne , Ronot Xavier , Adolphe Monique , Lechat Paul
Published: February 2005

The sodium salt of n‐butyric acid was found to inhibit the growth of asynchronous cultures of rabbit articular chondrocytes. This inhibitory effect was dose‐dependent between 1 mM and 5 mM, reversible, and accompanied by volume enhancement and modification of cellular morphology. Flow‐cytometric analysis showed that drug exposure led to a slowing‐down of the cell‐cycle progression; after 1 day's exposure, cells accumulated in G1, and after 2 or 3 days' treatment, in G2, without a blockage in M; the increase of cells in G2 was in fact due to an enhancement of binculeated cells. The treated cells had an increased RNA content. Articular chondrocytes seem to be target cells for sodium butyrate and therefore it represents a valuable biological tool for studying the mechanisms of their growth regulation.
Serum and substratum‐dependent coupled loss of differentiated and tumorigenic phenotypes in B16‐conv melanoma cells
Authors: Sato Seiji , Yamamoto Hiroaki , Yonezawa Minoru , Takeuchi Takuji
Published: February 2005

Clonal B16 mouse melanoma conv cells are tumorigenic spindle‐shaped cells (S‐type cells) exhibiting tyrosinase activity and melanosomes under usual culture conditions. When the cells passaged on glass substratum were plated for colony formation on plastic substratum in Eagle's minimum essential medium (MEM) with 10% calf bovine serum, most of them converted to fibroblastlike cells (F‐type cells) with the coupled loss of differentiated and tumorigenic phenotypes. However, they continued to be S‐type cells provided that they were plated on glass substratum. The conversion from S‐ to F‐type cell was not induced with high frequency even on plastic substratum when the concentration of calf serum in the medium was low (1–2%). These results indicate that both plastic substratum and serum factor are requisites for converting the phenotypic expression of the conv cells. Partial characterization of the serum factor indicates that it is adsorbable to plastic substratum, inactivated at 70°C for 10 min, salted out at 40% of saturated ammonium sulfate; in addition the factor seems to act on cells within 1 day after plating.
Junctional competence in clones of mammary epithelial cells, and modulation by conditioned medium
Authors: Stoker Michael
Published: February 2005

Colony‐forming epithelial cells exfoliated in human milk have been examined by immunofluorescence using antibodies to cytokeratins (tonofilaments), and to high molecular weight desmosomal core proteins. The cells may be classified by their ability to form junctional complexes with their neighbours. Those deficient in desmosomal junctions, called D − cells, grow into colonies of noncontiguous cells without desmosomes, and with a perinuclear network arrangement of cytokeratins. Junction forming, or D + cells, grow as contiguous cell sheets with abundant desmosomes and well developed bundles of tonofilaments. D − cells may also segregate D + cells among their progeny yielding mixed clones, and a gradual increase in the overall number of D + cells during culture. Established D + cells have surface markers characteristic of mammary epithelium and are presumably derived by exfoliation of luminal cells of the alveoli or ducts which contain desmosomal junctions. D − cells also possess mammary epithelial cell markers, but their origin is unknown. Medium conditioned by the Nil 8 line of hamster cells contains a junction‐promoting activity that accelerates the rate, or frequency, of segregation of D + cells from D − cells, so that milk cells grown in this medium predominently give closed colonies of D + cells. Medium conditioned by the MRC5 strain of human embryo lung cells, however, contains a junction‐inhibiting activity, which prevents new junction formation and probably destroys existing junctions, so that cells in this medium mostly grow as open colonies of cells with D − phenotype. It is hoped that studies with this experimental system will assist in the better understanding of normal and abnormal regulation of desmosomal junctions and their role in tissue integrity.
Cytosolic calcium, calcium fluxes, and regulation of alveolar macrophage superoxide anion production
Authors: Stickle Douglas F. , Daniele Ronald P. , Holian Andrij
Published: February 2005

The recently available compound quin‐2, which acts as a high affinity fluorescent indicator for calcium in the cytosol, was used to examine the role of calcium mobilization in the alveolar macrophage during the stimulation of 0 production by the tripeptide N‐formyl norleucyl leucyl phenylalanine (FNLLP). After preloading with quin‐2, the production of 0 was measured in conjunction with the transfer of 45Ca+2 and changes in quin‐2 fluorescence upon stimultion with FNLLP. When cells were maintained in low (10 μM) extracellular calcium medium the presence of 1.5 mM quin‐2 in the cytosolic space partially inhibited the rate of 0 production upon stimulation by FNLLP. Addition of 1 mM Ca+2 to the medium prior to stimulation rapidly restored the cell's capability to produce 0 upon stimulation at rates equal to control and extended the duration of stimulated 0 production as well. Quin‐2 fluorescence measurements indicated an increase in cytosolic Ca+2 upon stimulation with FNLLP. This increase was lowest under conditions in which 0 production was inhibited. The addition of 1 mM Ca+2 to the medium caused by itself a rapid but transient increase in cytosolic Ca+2 as measured with quin‐2 without stimulating 0 production. This intracellularly redistributed calcium was determined to be the source of the greater increase in cytosolic calcium during stimulation in the presence of high extracellular calcium. Measurements of 45Ca+2 transfer demonstrated a buffering of cytosolic Ca+2 changes by quin‐2, which in low calcium medium could deplete calcium stores. It is suggested that this effect, prior to stimulation, was responsible for the mitigated 0 response for those cells maintained in low calcium medium, wherein calcium stores could not be replenished. These results suggested that the cell's mechanism for regulating cytosolic and bound calcium concentrations may also play an integral role in its normal mechanism for stimulated 0 production. They further support the postulate that the commonly observed rise in the concentration of calcium in the cytosol upon formyl peptide stimulation is a concomitant but nonregulatory event only.
Maturation involves suppression of voltage‐gated currents in the frog oocyte
Authors: Taglietti Vanni , Tanzi Franco , Romero Rossana , Simoncini Luciana
Published: February 2005

Voltage‐ and time‐dependent currents having slow kinetics have been studied in plasma membranes of immature oocytes of the european frog, Rana esculenta. Ik, corresponding to an outward flow of K+, is activated at potentials more positive than about −40 mV, and subserves outward rectification; llr, corresponding to an outward flow of Cl−, is activated at potentials more negative than about −80 mV and subserves inward rectification. Such currents can act as negative feedback mechanisms in the control of membrane potential in the immature oocyte and limit to a somewhat restricted range its possible deviations from resting values. Besides lk, membrane depolarizations to potentials more positive than about + 30 mV are capable of activating lNa, corresponding to outflow of Na+. By contrast, the frog mature egg‐cell has a single voltage‐ and time‐dependent current, lM, activated at potentials more positive than +30 mV, with properties similar to lNa. The disappearance of lk and llr along with remarkable reduction in leakage lowers impedance in the egg membrane. It seems reasonable to suggest that the observed changes in membrane permeability reflect changes which have taken place along the maturation process and are of importance for successful fertilization.
The response of several murine embryonal carcinoma cell lines to stimulation of differentiation by α‐difluoromethylornithine
Authors: Schindler Joel , Kelly Michael , McCann Peter P.
Published: February 2005

In an attempt to better establish the relationship between polyamine levels and the differentiation of embryonal carcinoma cells, we have examined the ability of α‐difluoromethylornithine (DFMO), a known inducer of differentiation in one embryonal carcinoma cell line, to stimulate the differentiation of embryonal carcinoma cells from a variety of cell lines. Differentiation was monitored using a variety of criteria including morphological alterations and changes in biochemical and antigenic parameters. Depend ng on their response to difluoromethylornithine, three classes of cell lines could be identified, those which (1) differentiate extensively, (2) differentiate poorly, and (3) fail to differentiate. Three different classes of embryonal carcinoma cell lines reflect differential changes in polyamine levels resulting from inhibition of ornithine decarboxylase enzyme activity by DFMO. The specific cell lines which exhibit large decreases in both ornithine decarboxylase activity and polyamine levels also show extensive differentiation. The cell lines which show only moderate decreases in enzyme activity and polyamines differentiate poorly while the cell lines which fail to respond to DFMO in that polyamines do not drop below the threshold level necessary to induce differentiation fail to differentiate. These studies suggest that decreases in intracellular polyamines induce EC cell differentiation in vitro.
Reduced glutathione in chinese hamster ovary cells protects against inactivation of 3‐hydroxy‐3‐methylglutaryl Coenzyme A reductase by 2‐mercaptoethanol disulfide
Authors: Dotan Iris , Shechter Ishaiahu
Published: February 2005

When the disulfide of 2‐mercaptoethanol (ESSE) is added to the medium of cultured Chinese hamster ovary (CHO) cells, a time and concentration dependent release of 2‐mercaptoethanol to the medium is observed. The reduction of ESSE to 2‐mercaptoethanol by cells is a saturable process, the rate being approximately 50 nmoles of 2‐mercaptoethanol per mg cell protein for an hour upon exposure to 250 μM ESSE. Reduction rate of ESSE by cells attached to a substratum is independent of glucose and insulin for periods up to 4 hours. However, in detached cells, swirled in suspension, addition of glucose and insulin is necessary in order to obtain a linear reduction rate of ESSE. The rate limiting enzyme in the sterol biosynthetic pathway, 3‐hydroxy‐3‐methyl‐glutaryl Coenzyme A reductase (E.C. 1.1.1.34), is inhibited by ESSE when isolated from CHO cells but total nonsaponifiable lipids synthesis from [2−14C]‐acetate in intact cells is not affected by ESSE at concentrations up to 500 μM. Cytosolic reduced glutathione can spontaneously exchange disulfide bonds with ESSE and thus prevent it from inhibiting the reductase. Cultured cells respond to ESSE administration by elevating their total and acid‐soluble glutathione levels. The use of ESSE as a perturbant of the GSH Status in cells is discussed.
Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT‐29 selected for growth in sugar‐free medium and its inhibition by glucose
Authors: Zweibaum Alain , Pinto Moïse , Chevalier Guillemette , Dussaulx Elisabeth , Triadou Nicole , Lacroix Brigitte , Haffen Katy , Brun Jean‐Louis , Rousset Monique
Published: February 2005

In order to study the effect of glucose on the differentiation of cultured human colon cancer cells, a subpopulation of HT‐29 cells was selected for its capacity to grow in the total absence of sugar. These cells (GIc−cells) exhibit, after confluency, an enterocytic differentiation, in contrast to cells grown with glucose (Glc+ cells), which always remain undifferentiated. The differentiation is characterized by a polarization of the cell layer with apical brush borders and tight junctions, and by the presence of sucrase‐isomaltase. The differentiation of Glc−cells is reversible: the addition of glucose to postcon‐fluent cultures of Glc− cells results in an inhibiting effect on the expression of sucrase‐isomaltase; switching growing cultures of Glc−cells to the Glc+ medium for several passages results in a progressive reversion to the undifferentiated state, which is completed after seven passages. The dedifferentiation process is associated with a parallel, passage‐related, increase in the rates of glucose consumption and lactic acid production, and decreases of intracellular glycogen content, which return to the values of the undifferentiated original Glc+ cells. The values of these metabolic parameters are correlated, at each passage, with the degree of dedifferentiation of the cells. When these dedifferentiated cells, after having been cultured in Glc+ medium for 20 passages, are switched back to the Glc− medium, they readily grow without mortality, and reexpress the same enterocytic differentiation as the parent Glc− cells. These results show that the capacity of this subpopulation to grow and differentiate in the absence of sugar is a stable characteristic. They further suggest that glucose metabolism interferes with the program of differentiation of HT‐29 cells.
Forskolin induction of S‐100 protein in glioma and hybrid cells
Authors: Higashida Haruhiro , Sano Mamoru , Kato Kanefusa
Published: February 2005

The S‐100 protein level in mouse neuroblastoma (N18TG‐2 and NIE‐115), rat glioma (C6, C6BU‐1, and C6V‐1), and hybrid (NG108‐15, 140‐3, 141‐B, NBr10A, NBr20A, NCB20, and NX3IT) cells was determined with a sensitive enzyme immunoassay system that uses a rabbit antibody to bovine brain S‐100 protein. S‐100 protein was detected in glioma but not in neuroblastoma cells. All seven hybrid cells derived from neuroblastoma and glioma or other types of cells were found to possess a very little or undetectable S‐100 protein. The induction of S‐100 protein level in prestationary phase cultures of glioma C6BU‐1 cells was examined by forskolin, which was a highly specific activator of adenylate cyclase of the cells and produced morphological differentiation. After incubation with 10 μM forskolin for 48 hr, the S‐100 protein level increased 2–2.5‐fold in C6BU‐1 glioma cells whose mean control level was 60 ± 26 ng/mg protein (± SD). The forskolin induction of S‐100 protein in the cells was dose dependent, and the concentration of forskolin required for 50% activation of S‐100 protein was about 0.6 μM. The increase by forskolin was initiated from 10–15 hr after incubation with it and was inhibited with cycloheximide and actinomycin D. In NG108‐15 hybrid cells the induction of S‐100 protein was also observed by forskolin as well as prostaglandin (PG) E1 plus theophylline which are known to activate adenylate cyclase of the cells. The results indicate that S‐100 protein biosynthesis is genetically controlled in these clonal cells, and that S‐100 protein can be regulated in a cAMP‐dependent fashion in prestationary cultures.
Transient and sustained effects of hormones and calcium on membrane potential in a bone cell clone
Authors: Ferrier Jack , Illeman Alan , Zakshek Eva
Published: February 2005

Measurements were made of the electrophysiological and cAMP response to changes in extracellular [Ca2+] and to hormone application in a bone cell clone. Both transient and long‐term electrophysiological responses were studied. An increase in extracellular [Ca2+] usually resulted in a transient hyperpolarization of about 60‐sec duration. In addition, increases in extracelllular [Ca2+] from 0.9 to 1.8 mM and from 1.8 to 3.6 mM resulted in long‐term hyperpolarization and increased potential fluctuations. Increasing bathing [Ca2+] until the membrane potential reached the K+ equilibrium level resulted in a significant decrease in fluctuations. Addition to the bathing medium of quinine, a putative blocker of the Ca2+‐dependent K+ channel, resulted in long‐term depolarization of the mean membrane potential, and a long‐term decrease in potential fluctuations. Addition of Mg2+, a mild antagonist of Ca2+ entry into the cell, produced transient depolarization and reduction of potential fluctuations. These effects suggest that the potential fluctuations reflect cytoplasmic [Ca2+] fluctuations via Ca2+‐dependent K+membrane channels. Under an extracellular[Ca2+] of 1.8 mM, the application of prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone produced no significant effect on mean membrane potential or on the sustained potential fluctuations, but PGE2 did significantly raise intracellular cAMP. Under an increased bathing [Ca2+], significant changes in mean potential and fluctuations did occur in response to PGE2, but not in response to the other hormones, while the PGE2 effect on cAMP was not greatly changed. Hyperpolarizing transients of about 30‐sec duration occurred in response to all of the hormones, particularly at an extracellular [Ca2+] of 3.6 mM. Thus, there are both transient and long‐term electrophysiological responses to hormone application, with only the long‐term response correlated with the production of cAMP. These electrophysiological responses may represent separate transient and long‐term calcium transport responses to hormone application.
Dexamethasone and retinyl acetate similarly inhibit and stimulate EGF‐or insulin‐induced proliferation of prostatic epithelium
Authors: Chaproniere Donna M. , Webber Mukta M.
Published: February 2005

Prostatic epithelium proliferates in a defined medium consisting of basal medium RPMI1640 containing transferring (1 μg/ml), EGF (10 ng/ml), and insulin (3.7 μg/ml or 0.1 lU/ml). Although neither dexamethasone nor retinyl acetate affected the proliferation of prostatic epithelium in RPMI1640 containing trans‐ferrin alone, they modify the mitogenic effect of EGF and insulin. Dexamethasone at 10−10 M or retinyl acetate at about 3 × 10−9 M inhibits proliferation stimulated by EGF. Higher concentrations of dexamethasone (10−8−10−6 M) or retinyl acetate (3 × 10−8−10−7 M) enhance the mitogenic activity of EGF. Dexamethasone had a similar effect in the presence of insulin. However, retinyl acetate stimulated, but did not significantly inhibit, proliferation in the presence of insulin. These results suggest that both dexamethasone and retinyl acetate, and possibly other glucocorticoids and retinoids, may regulate the proliferation of prostate epithelium by a dose‐dependent modification of the activity of insulin and EGF.
Relationship of finite proliferative lifespan, senescence, and quiescence in human cells
Authors: Stein Gretchen H. , Namba Masayoshi , Corsaro Cheryl M.
Published: February 2005

Cell hybrids were formed between human diploid fibroblasts (HDF) and carcinogen‐transformed HDF to determine the relationship among: (1) finite proliferative lifespan, which we define as an age‐related failure of a population to achieve one population doubling in 4 weeks; (2) arrest in a senescent state, which we define as cessation of DNA synthesis in a viable culture that is at the end of its lifespan by the above definition; and (3) arrest in a quiescent state, which we define as cessation of DMA synthesis in a young culture that is crowded or mitogen‐deprived. HDF express all three of these phenotypes, which we have abbreviated FPL+, S+, and Q+, respectively. Carcinogen‐transformed HDF are transformed to immortality (FPL−) and inability to achieve quiescence (Q−). They have no S phenotype because, by definition, this phenotype only exists in FPL+ cells. Fusion of FPL+, Q+, S+ HDF × FPL−, Q− carcinogen‐transformed HDF produced hybrid clones that were FPL+, Q−, and S−, where the S− phenotype means that individual cells continued to synthesize DNA in cultures that had reached the end of their lifespan by our definition. These results are consistent with our hypothesis that senescent HDF and quiescent HDF may share a common mechanism for arrest in G1 phase. We have suggested that this could occur if the aging mechanism that is responsible for the FPL+ phenotype is a progressive decrease in the ability of cells to recognize or respond to mitogenic growth factors. If so, then cells would become physiologically mitogen‐deprived at the end of their lifespan, which would cause them to arrest in the senescent state by the same mechanism that causes young cells to arrest in the quiescent state when they are mitogen‐deprived. This hypothesis predicts that the FPL+ phenotype can be separated from the S+ phenotype–i.e., FPL+ cells can be S+ or S− –and that the Q and S phenotypes are linked‐i.e., FPL+ cells are either Q+ and S+ or Q− and S−. Both these predictions are supported by the present data.
Stimulation of hexose uptake by haemopoietic cell growth factor occurs in WEHI‐3B myelomonocytic leukaemia cells: A possible mechanism for loss of growth control
Authors: Whetton A. D. , Bazill G. W. , Dexter T. M.
Published: February 2005

WEHI‐3B myelomonocytic leukaemia cells secrete a haemopoietic cell growth factor (HCGF) which facilitates the proliferation and development of multipotential stem cells and committed progenitor cells. Several cloned, nonleukaemic cell lines (FDC‐P cells) are absolutely dependent on HCGF and die in the absence of it. In these cell lines, factor dependence is associated with the ability of HCGF to increase glucose uptake, thereby controlling glycolytic flux and intracellular ATP levels. We have now investigated the effects of HCGF on glucose uptake in WEHI‐3B cells. At 20°C 2‐deoxyglucose uptake could be stimulated by the addition of HCGF to the extracellular medium. L‐glucose uptake was markedly lower than 2‐deoxyglucose uptake and did not respond to the addition of HCGF. At 37°C no HCGF stimulation of 2‐deoxyglucose uptake was found. However, at this temperature HCGF release from WEHI‐3B cells was markedly higher than at 20°C. Our experiments indicate that HCGF stimulates the glucose transport system in both WEHI‐3 cells and FDC‐P cells. The similarities between the WEHI‐3B cell and FDC‐P2 cell polypeptide phenotype were investigated using two‐dimensional isoelectric focussing/poly‐acrylamide gel electrophoresis. This revealed a high degree of correlation between the two cell types in their protein constituents, indicating a close relationship between the normal and leukaemic cells. These similarities between WEHI‐3B cells and FDC‐P2 cells are considered and their relevance to haemopoiesis and leukaemogenesis is discussed.
The deoxyglucose method adapted for studies of glucose metabolism in the early chick embryo
Authors: Baroffio Anne , Kucera Pavel
Published: February 2005

14C‐2‐deoxyglucose (DG), currently employed in in vivo studies of brain glucose metabolism, has been used for determination of glucose consumption in the in vitro developing chick embryo. DG, presented in traces, accumulates in the embryo in proportion with incubation time. Analysis of tissue homogenates shows that the accumulated radioactivity is due to both phosphorylated (DGP) and nonphosphorylated DG. As it is only the radioactivity originating from the DGP that is proportional to glucose utilization, the nonphosphorylated DG must be washed out. The washout shows two distinct kinetics: a fast one corresponding to DG that has entered the cells but has not yet been phosphorylated and a slow one that is probably due to a dephosphorylated DGP coming from a different cellular compartment. On the basis of these results the optimal experimental conditions have been defined, allowing quantitative studies of glucose metabolism during the first day of development of the chicken embryo. From 18 to 24 hr of incubation (end of gastrulation), total glucose consumption increases from 50 nmol · h−1 at stage 3–4 to 90 nmol · h−1 at stage 6–7. This increase mainly reflects the growth of the blastodisc. Comparison with the values of O2 uptake measured at the same period of development suggests that only a fraction of the glucose consumed is oxidized, the major part being converted aerobically to lactate.
Regulation of cell proliferation inhibitory and stimulatory factors diffused by 3T3 cultured cells
Authors: Harel Louise , Blat Christiane , Chatelain Gilles
Published: February 2005

The growth rate of normal cells multiplied in vitro decreases as the cell density of the culture increases. Previous results suggested that this density‐dependent inhibition of growth in nontransformed cells was due to the diffusion of growth inhibitory substances in the medium of dense cultures. In this paper, we demonstrate that dense cultures of 3T3 cells secrete inhibitory and stimulatory factors. Macromolecules of conditioned medium were fractionated on Biogel P150 and the different fractions were tested on quiescent cultures of 3T3 cells stimulated or not to proliferate by addition of α globulin. When target cells were not stimulated to proliferate by addition of exocrine growth factors, we observed the inhibitory activity of a large molecular weight inhibitor (IDF45) and the stimulatory activity of autocrine growth factors (fraction about 35 and 10 K molecular weight), on the incorporation of 14C inosine into nucleotide pool and RNA. However, DNA synthesis was significantly stimulated with fraction 10 K only. This discrepancy between the stimulation of RNA and DNA synthesis may be explained by the presence, simultaneously, of inhibitory and stimulatory factors in fraction 35 and 10 K molecular weight. The presence of inhibitory factor was demonstrated when the fractions were tested on target cells stimulated to proliferate by α globulin addition and labeled with 14C thymidine. In these conditions, the stimulatory activity of autocrine growth factors was not observable, and only the inhibitory activity on DNA synthesis of fractions 35 and 10 K appeared. It is tempting to assume that the regulation of in vitro cell proliferation is determined by the balance between these antagonist stimulatory and inhibitory autocrine growth factors.
Estrogen receptor expression in serially cultivated rat endometrial cells: Stimulation by forskolin and cholera toxin
Authors: Heimann Ruth , Rice Robert H. , Gross Mary K. , Coe Eleanor L.
Published: February 2005

Serially propagated with 3T3 feeder layer support, epithelial cells derived from normal rat endometrium expressed estrogen receptor activity. Specific binding of 17‐β‐estradiol was in the range of 30–60 fmol/mg of protein and was of high affinity (Kd = 0.3 nM). A survey of cell lines derived from several other normal epithelia showed that rat vaginal and human cervical cultures also had high‐affinity estrogen receptors (6–13 fmol/mg of protein), while rat epidermal and esophageal cells had no detectable activity. In the endometrial cultures, receptor levels were elevated nearly two‐ to fourfold by cholera toxin or forskolin in the medium. This effect was detectable after 4 hr but not 1 hr of treatment and did not occur in the presence of cycloheximide. We conclude that serially cultivated rat endometrial cells retain hormonal properties expressed in vivo while exhibiting some keratinocyte character. These cells may provide a useful model for study of receptor modulation.
An approach to a selection system for adenosine‐deaminase‐positive (ADA+) cells and detection of rat ADA+ “revertants”
Authors: Hischhorn Rochelle , Ellenbogen Amy , Martiniuk Frank
Published: February 2005

We have substituted deoxyadenosine or adenosine for hypoxanthine in the standard HAT selection system in an attempt to select for ADA‐normal (ADA+) cells. ADA− human lymphoid line cells could not utilize deoxyadenosine as an alternative to hypoxanthine as a purine source (DAT) and failed to grow but were only somewhat inhibited in growth when adenosine was substituted for hypoxanthine (AAT). In contrast, ADA+ cells utilized adenosine or deoxy‐adenosine as efficiently as hypoxanthine as a purine source. Growth in DAT, but not in HAT, of an artificial mixture of one ADA+ human lymphoid cells in 1,000 ADA− cells resulted in enrichment of ADA+ cells to 25–86% of total cells. When we grew a rat ADA− cell line in two variations of the DAT system, we detected at least three electrophoretically different ADA+ patterns, one of which corresponded to normal rat ADA. These could represent “revertants.”
Abortive transformation of temperature‐sensitive mutants of rat 3Y1 cells by simian virus 40: Effect of cellular arrest states on entry into S phase and cellular proliferation
Authors: Mitsudomi Tetsuya , Kimura Genki
Published: February 2005

Four temperature‐sensitive (ts) mutants of rat 3Y1 fibroblasts, representing independent complementation groups, cease to proliferate predominantly with a 2n DNA content, at the restrictive temperature (39.8°C)(temperature arrest) or at the permissive temperature (33.8°C) at a confluent cell density (density arrest) (Ohno et al., 1984). We studied the temperature‐ or the density‐arrested cells of these mutants infected with simian virus 40 (SV40) or its mutants affecting large T or small t antigen with respect to kinetics at 39.8°C of entry into S phase and cellular proliferation. Three mutants, 3Y1tsD123, 3Y1tsF121 and 3Y1tsG125, expressed T antigen and entered S phase at 39.8°C from both the arrested states after infection with either wild‐type, tsA mutants, or a .54/.59 deletion mutant of SV40, whereas in the density‐arrested 3Y1tsH203, expression of T antigen and entry into S phase were inefficient and ts. Following the WT‐SV40 induced entry into S phase, the temperature‐arrested 3Y1tsD123 detached from the substratum with no detectable increase in cell number, whereas the density‐arrested ones completed a round of the cell cycle and then detached. 3Y1tsF121 and 3Y1tsG125 in the both arrested states proliferated through more than one generation. 3Y1tsF121 and 3Y1tsG125 in the density‐arrested state infected with tsA mutants once proliferated and then ceased to increase in number as the percentage of T‐antigen positive population decreased. These results suggest that wild‐type and tsA‐mutated large T antigens are able to overcome the cellular ts blocks of entry into S phase in the 3 ts mutants of 3Y1 cells in both the arrested states, and that small t antigen is not required to overcome the blocks. It is also suggested that cellular behaviors subsequent to S phase (viability, mitosis, and proliferation in the following generations) depend on cellular arrest states, on traits of cellular ts defects, and on the duration of large T antigen expression.
Defective endocytosis of low‐density lipoprotein in monensin‐resistant mutants of the mouse Balb/3T3 cell line
Authors: Tomita Kichinobu , Ono Mayumi , Masuda Akinori , Akiyama Shin‐Ichi , Kuwano Michihiko
Published: February 2005

Two monensin‐resistant clones show similar low‐density lipoprotein binding activity but less internalization or degradation of low‐density lipoerotein than the parental Balb/3T3 or other resistant clone. Sterol synthesis from radioactive acetate in the resistant mutant, MO‐5, is inhibited by more than 70% of control in the presence of tenfold higher amounts of low‐density lipoprotein than the dose that inhibits the parental Balb/3T3 to similar level. 3‐Hydroxy‐3‐methylglutaryl coenzyme A reductase activity of Balb/3T3 and MO‐5 is inhibited by 48% and 27% of control, respectively, in the presence of 10 μg/ml of low‐density lipoprotein. Colloidal silica gradient centrifugation shows that transport of low‐density lipoprotein from the surface membrane to the lysosome is much slower in MO‐5 cells than in Balb/3T3 cells. Down regulation of low‐density lipoprotein receptors on the cell surface in Balb/3T3 is observed by exposing the cells to 5–15 μg/ml low‐density lipoprotein, whereas only slight if any down regulation is observed when MO‐5 cells are treated with low‐density lipoprotein. The altered endocytosis of low‐density lipoprotein behaves as a dominant trait in hybrids of MO‐5 and THO2‐2, a derivative of Balb/3T3 resistant to both ouabain and 6‐thioguanine.
Lipoproteins increase growth of mitogen‐stimulated arterial smooth muscle cells
Authors: Libby Peter , Miao Paul , Ordovas Jose M. , Schaefer Ernst J.
Published: February 2005

The relationship between lipoproteins and growth of aortic smooth muscle cells has been a matter of controversy. We therefore reexamined this issue using serum‐free defined media methodology. By themselves, LDL or HDL (50–500 μg/ml) from normolipemic human or bovine plasma produced little or no growth of homologous aortic smooth muscle cells incubated in serumfree medium that was supplemented with insulin and transferrin to maintain cell viability. In fact, LDL prepared in the absence of an antioxidant (BHT) was toxic to these cells. However, in the presence of maximally effective concentrations of platelet‐derived growth factor (PDGF), LDL or HDL consistently increased the growth of homologous smooth muscle cells (up to twofold increases in DNA accumulation in 48 hr). Lipoproteins also augmented the growth response of arterial smooth muscle cells to fibroblast growth factor or epidermal growth factor. The mechanism of this effect was investigated further with HDL, because, in contrast to LDL, HDL apoproteins are water‐soluble. Neither HDL delipidated by solvent extraction (apoHDL), purified bovine apoA‐I, nor cholesterol added in the form of phospholipid vesicles appreciably increased PDGF‐induced growth of bovine smooth muscle cells. However, HDL‐like particles reconstituted by sonication of apoHDL with cholesterol and phospholipids did increase the growth of cultures of bovine smooth muscle cells treated with PDGF. Uptake of tritiated thymidine by cultures incubated with partially purified PDGF alone (10 μg/ml) was 5,693 ± 235 dpm/24 hr compared to 10,381 ± 645 dpm/24 hr (p < 0.01) in the presence of both PDGF and reconstituted HDL‐like particles (250 μg protein/ml). Thus both the lipid and protein components of HDL may be necessary for optimal potentiation of growth of mitogen‐stimulated cells. These results indicate that lipoproteins from normolipemic sera are not bona fide growth factors but can potentiate the growth of mitogen‐stimulated cells, perhaps by supplying exogenous cholesterol required for membrane biogenesis. This finding might be important in arterial injury when the release of PDGF and exposure to plasma lipoproteins could act in concert to stimulate the proliferation of smooth muscle cells.
The fate of DNA originally existing in the zygote nucleus during achromosomal cleavage of fertilized echinoderm eggs in the presence of aphidicolin: Microscopic studies with anti‐DNA antibody
Authors: Yamada Hisafumi , Hirai Setsuro , Ikegami Susumu , Kawarada Yoshihiko , Okuhara Eiji , Nagano Hiroshi
Published: February 2005

In fertilized echinoderm eggs, the male and female pronuclei fuse to form the diploid nucleus even in the presence of aphidicolin, a specific inhibitor of eukaryotic DNA polymerase‐α. The subsequent first cleavage is independent of chromosomes but dependent on spindle and amphiaster. The fate of DNA originally existing in the fused nucleus during achromosomal cleavage of fertilized sea urchin and starfish eggs induced by aphidicolin was determined using antidenatured DNA antibody. The nucleus is not formed in the divided daughter cells at the two‐cell stage but the nuclear‐envelope‐free chromatin mass which is unassociated with mitotic apparatus remains in the center region of embryos, especially near the first cleavage furrow. These results indicate that the condensed and nonreplicated chromatin can not be associated with spindle and asters in the presence of aphidicolin.