bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) on the growth inhibitory and increased phosphatidylinositol (PI) responses induced by epidermal growth factor (EGF) in A431 cells
Authors: Smith Kathryn B. , Losonczy Ilona , Sahai Atul , Pannerselvam Mounanandham , Fehnel Paula , Salomon David S.
Published: February 2005

Epidermal growth factor (EGP) inhibited the growth of A431 human epidermoid carcinoma cells. The tumor promoting, phorbol ester, 12‐O‐tetradeca‐noylphorbol‐13‐acetate (TPA) also retarded A431 cell growth. Addition of both TPA and ECF inhibited cell growth in an additive or synergistic manner depending upon the initial plating density of the cultures. EGF increased the production of diacylglycerol (60–70%) and stimulated the synthesis of phosphatidylinositol (PI) from 3H‐inositol (three‐ to fourfold increase). Both of these responses were attenuated in the presence of TPA. TPA alone stimulated the production of diacylglycerol (DG) but had little effect on PI synthesis. The biological effect of TPA appeared to be mediated by the presence of a high‐affinity receptor for phorbol esters on A431 cells. Moreover, the binding of 125I‐EGF to A431 cells was unaffected by TPA, suggesting that the antagonistic effects of TPA were occurring distal to the EGF receptor. These findings also indicated that although TPA and EGF both inhibited A431 cell growth, this effect could be dissociated from changes in PI synthesis but may be dependent upon transient changes in DG production.
Increase of latent HMG‐COA reductase activity with increasing density of cell cultures
Authors: Chen Harry W.
Published: February 2005

The total (active plus latent) activity of HMG‐CoA reductase declined linearly with increasing cell density in cultures of three lines of mammalian cells. The active form disappeared almost entirely under this condition, while the latent (presumably phosphorylated) form increased to some extent. The disappearance of active HMG‐CoA reductase with concomitant increase in the proportion of latent HMG‐CoA reductase was correlated with the decline in cellular multiplication and sterol synthesis. These results suggest that interconversion of HMG‐CoA reductase between active and inactive forms through phospho‐rylation‐dephosphorylation can be associated with changes in the rate of cellular proliferation in cell cultures. However, the decreased rate of sterol synthesis followed more closely the slower disappearance of the total HMG‐CoA reductase activity than the rapid decrease of the active form of the reductase alone. Therefore, changes in the rate of cellular proliferation can affect the interconversion of HMG‐CoA reductase between active and inactive forms through reversible phosphorylation. However, phosphorylation of the enzyme to the inactive form appears not to be the mechanism by which the sterol synthetic rate is regulated in confluent cell cultures. Rather, the amount of total HMG‐CoA reductase determines the rate of sterol synthesis.
Two populations of acid hydrolase‐containing particles in rat epididymis
Authors: Mayorga Luis , Bertini Francisco
Published: February 2005

Two populations of acid hydrolase‐containing particles were distinguished in homogenates of rat epididymis. One of them was rich in acid phosphatase activity, equilibrated at density 1.17 in a sucrose gradient, and it sedimented between 12,000g 2.5 min and 43,000g 60 min (light particles). The other was poor in acid phosphphatase activity and rich in N‐acetyl‐β‐D‐glucosaminidase, arylsulphatase, and β‐glucuronidase activity, equilibrated at density 1.20 in a sucrose gradient and it sedimented between 400g 2.5 min and 12,000g 2.5 min (heavy particles). 131I‐albumin (RISA) injected into the lumen of the cauda was partially recovered in subcellular particles of homogenates of this region. These particles, incubated at pH 5, were able to digest the engulfed RISA. The subcellular distribution of RISA‐containing particles and RISA‐digesting particles was similar to that of the heavy hydrolase‐containing particles. This suggests that these latter are engaged, at least in part, in heterophagic processes.
Efficient differentiation of proadipocyte stem cells on nonadherent surfaces: Evidence for differentiation without DNA synthesis
Authors: Yun Kankatsu , Hoerl Bryan J. , Scott Robert E.
Published: February 2005

The differentiation of low density BALB/3T3 T proadipocytes that are cultured in standard tissue culture flasks can be induced by heparinized medium containing human plasma. It has been shown that under these conditions, cells first growth‐arrest at a distinct state in the G1 phase of the cell cycle, designated GD, and thereafter differentiate within 8 to 12 days. In this paper, we report that the kinetics of proadipocyte differentiation can be significantly accelerated by culture of cells in differentiation‐promoting medium on non‐adherent surfaces, such as agarose‐coated plates or bacteriological Petri dishes. Data also show that in a nonadherent microenvironment extensive differentiation can occur in the absence of DNA synthesis. This was established most convincingly by the demonstration that placement of mitotic cells in heparinized medium containing human plasma and hydroxyurea on agarose‐coated Petri dishes induced 70–80% of the cells to GD arrest and differentiate without traversing the S phase of the cell cycle. It is concluded that under appropriate microenvironmental conditions metabolic events that occur solely in the late M or early G1 phase of the cell cycle can mediate the integrated control of proadipocyte proliferation and differentiation.
Proliferative kinetics and differentiation of murine blast cell colonies in culture: Evidence for variable G0 periods and constant doubling rates of early pluripotent hemopoietic progenitors
Authors: Suda Tohio , Suda Junko , Ogawa Makio
Published: February 2005

Several investigators have described hemopoietic colonies expressing multilineage differentiation in culture. We recently identified a class of murine hemopoletic progenitors which form blast cell colonies with very high replating efficiencies. In order to clarify further the relationship between progenitors for blast cell colonies and progenitors for the multilineage hemopoietic colonies in culture, we carried out analyses of kinetic and differentiation properties of murine blast cell colonies. Serial observations of the development of blast cell colonies into multilineage (and single lineage) colonies in cultures of spleen cells obtained from 5‐fluorouracil (5‐FU)‐treated mice confirmed the transitional nature of the murine blast cell colonies. The data also suggested that the early pluripotent progenitors are in G0 for variable periods, and that when triggered into cell cycle, they proliferate at relatively constant doubling rates during the early stages of differentiation. The notion that some of the pluripotent progenitors are in G0 was also supported by long‐term thymidine suicide studies in which spleen cells were exposed to 3H‐thymidine with high specific activity for 5 days in culture, washed, and assayed for surviving progenitors. Comparison of replating abilities of day‐7 and day‐16 blast cell colonies from normal as well as 5‐FU‐treated mice indicated that some of the day‐7 blast cell colonies are derived from maturer populations of progenitors which are sensitive to 5‐FU. In contrast, progenitors for the day‐16 blast cell colonies are dormant in cell cycle and were not affected by 5‐FU treatment. Previously we reported that progenitors for day‐16 blast cell colonies have a significant capacity for self‐renewal. These observations suggest the hypothesis that the capability for self‐renewal is accompanied by long periods of G0, and that once commitment to differentiation takes place, then active cell division occurs.
A quantitative analysis of the aging of human glial cells in culture
Authors: Pontén Jan , Stein Wilfred D. , Shall Sydney
Published: February 2005

The kinetics of aging of normal human diploid brain cells in culture have been determined using the miniclone technique in which cells are cloned in the presence of a large number of other cells. The miniclone technique records the behaviour of every viable cell in the sample, not merely those cells capable of forming visible clones. This technique permits the direct measurement of the reproductive potential of individual cells growing in buik culture and of the dispersion of the sizes of colonies generated by dividing cells. The fraction of cells that are able to divide declines smoothly and continuously from the beginning of in vitro cultures of human glial cells. There is a broad distribution of colony sizes; even at the earliest passages there are significant numbers of small colonies. With increasing age of the culture there is a shift in the distribution, so that fewer large colonies and more small colonies occur. The distribution of intermitotic times is almost identical in young and middle‐aged cultures. Our data seem to exclude quite positively any description in terms of a catastrophe or any abrupt change in the population. On the contrary, the decline in reproductive potential may be described adequately either as a linear change with time, or as predicted by the mortality theory of Shall and Stein (1979), in which the single constant, gamma, describes the change in reproductive potential over the entire lifetime.
Glycosaminoglycans synthesized by cultured bovine corneal endothelial cells
Authors: Robinson Julie , Gospodarowicz Denis
Published: February 2005

Bovine corneal endothelial (BCE) cells seeded and grown on plastic dishes were labeled with 35S‐sulfate or 3H‐glucosamine for 48 h at various phases of growth of the cultures. Newly synthesized proteoglycans were isolated from the culture medium and from the extracellular matrix (ECM) produced by the BCE cells, and the glycosaminoglycan (GAG) component of the proteoglycans was analyzed. Cells actively proliferating on plastic surfaces secreted an ECM that contained heparan sulfate as the major 35S‐labeled GAG (86%) and dermatan sulfate as a minor component (13%). Upon reaching confluence, the BCE cells incorporated 35S‐labeled chondroitin sulfate (20%), as well as heparan sulfate (66%) and dermatan sulfate (14%), into the ECM. Seven‐day postconfluent cells incorporated newly synthesized heparan sulfate and dermatan sulfate into the matrix in approximately equal proportions. Dermatan sulfate was the main 35S‐labeled GAG (60–65%) in the medium of both confluent and postconfluent cultures. 35S‐Labeled chondroitin sulfate (20–25%) and heparan sulfate (15%) were also secreted into the culture medium. The type of GAG incorporated into newly synthesized ECM was affected when BCE cells were seeded onto ECM‐coated dishes instead of plastic. BCE cells actively proliferating on ECM‐coated dishes incorporated newly synthesized heparan sulfate and dermatan sulfate into the ECM in a ratio that was very similar to the ratio of these GAGs in the underlying ECM. Addition of mitogens such as fibroblast growth factor (FGF) to the culture medium altered the type of GAG synthesized and incorporated into the ECM by BCE cells seeded onto ECM‐coated dishes if the cells were actively growing, but had no effect on postconfluent cultures.
Teratocarcinoma cells exhibit growth cooperativity in vitro
Authors: Isacke Clare M. , Jane Deller M.
Published: February 2005

Malignant PC13 embryonal carcinoma (EC) cells differentiate in vitro in response to retinoic acid, giving rise to a population of benign endoderm‐like cells (END), termed PC13 END. PC13 EC and PC13 END cells exhibit growth cooperatively in co‐culture, whereby the EC cells stimulate END cell proliferation and the END cells can support EC cell multiplication. The EC cells' stimulatory effect operates via soluble, diffusible factors which are also active on a range of fibroblast cell lines. END cells support the multiplication of EC cells plated at low density, via a multifactorial mechanism. Contact‐dependent effects can operate in the absence of END cell metabolic activity, while contact‐independent effects require the continuous presence of live END cells. It was observed that there was a variation in the ability of fibroblast cell lines to act as EC cell feeders. Similar interactive events may be important during the in vivo proliferation and differentiation of teratocarcinoma cells and their embryonic counterparts.
Hormonal regulation of discrete portions of the cell cycle: Commitment to DNA synthesis is commitment to cellular division
Authors: Wharton Walker
Published: February 2005

Density‐arrested human fibroblasts were stimulated to traverse G0/G1 and initiate DNA synthesis by the addition of medium containing either serum or a combination of platelet‐derived growth factor and platelet‐poor plasma. Medium containing a combination of epidermal growth factor and high concentrations of insulin also stimulated DNA synthesis in platelet factor‐treated quiescent cells. Platelet factor was required only to initiate proliferation. Epidermal growth factor and insulin then allowed G1 traverse and commitment to DNA synthesis. Cells could complete S, G2, and M in unsupplemented medium lacking peptide growth factors.
In vitro demonstration of deleterious effect of steel mutation on spermatogenesis in mice
Authors: Nishimune Yoshitake , Haneji Tatsuji , Maekawa Mamiko , Kitamura Yukihiko
Published: February 2005

The effect of the steel mutation on spermatogenesis was investigated by using organ culture. Cultured cryptorchid testes from C57BL/6− +/+ mice showed an effective differentiation of type A spermatogonia in response to an increased concentration of bovine serum. In contrast, the cryptorchid testes from C57BL/6 – S/d/ + mice showed only limited differentiation of type A spermatogonia even in the highest concentration of serum tested. The type A spermatogonia of mutant mice showed the normal mitotic response but did not differentiate further. The findings reported here are the first in vitro demonstration of the deleterious effects of the steel mutation on testicular germ cell differentiation and suggest that the effect of the steel mutation is expressed in Sertoli cells, the only supporting element in seminiferous tubules, or germ cells themselves.
In situ oxygen consumption rates of cells in V‐79 multicellular spheroids during growth
Authors: Freyer J. P. , Tustanoff E. , Franko A. J. , Sutherland R. M.
Published: February 2005

The rate of consumption of oxygen by V‐79 cells in multicellular spheroids was measured as a function of the spheroid diameter. In situ consumption was equal to that of exponentially growing cells for spheroids less than 200 μm in diameter. The rate of oxygen consumption decreased for cells in spheroids between 200 and 400 μm diameter to a value one‐fourth the initial, then remained constant with further spheroid growth. Comparison of consumption rates for spheroid‐derived cells before and after dissociation from the spheroid structure indicated that the spheroid microenvironment accounted for only 20% of the change in oxygen consumption rate. Cell‐cell contact, cell packing, and cell volume were not critical parameters. Plateau‐phase cells had a fivefold lower rate of oxygen consumption than exponential cells, and it is postulated that the spheroid quiescent cell population accounts for a large part of the intrinsic alteration in oxygen consumption of cells in spheroids. Some other mechanism must be involved in the regulation of cellular oxygen consumption in V‐79 spheroids to account for the remainder of the reduction observed in this system.
Selection of mouse macrophage‐like sublines that differ in leukemogenic potential and characterization
Authors: Kasukabe Takashi , Honma Yoshio , Hozumi Motoo
Published: February 2005

The murine macrophage‐like cell line (Mm‐1), which is nonleukemogenic to syngeneic SL mice, was originally derived from spontaneously differentiated cells of a clonal line of mouse myeloid leukemia cells (M1). In the present experiment, variant cell lines with a high (Mm‐A), moderate (Mm‐P), and little or no (Mm‐Sl and Mm‐S2) leukemogenic potential were obtained from the Mm‐1 cells. The mean survival times of syngeneic SL mice inoculated i.p. with 5 × 106 Mm‐A and Mm‐P cells were 17 and 33 days, respectively, whereas almost all the mice inoculated with Mm‐S1 or Mm‐S2 cells survived for more than 90 days. These variant cell lines did not lose their macrophage‐like characteristics in vitro. These variant cell lines phagocytized latex beads and sensitized sheep erythrocytes, produced lysozyme, and adhered to culture dishes. The four variant cell lines showed no significant difference in porliferation rates in vitro in liquid medium containing 10% calf serum, but Mm‐A cells could grow both in soft agar medium in the absence of ascitic fluid containing colony‐stimulating factor (CSF) and in liquid medium containing 1% serum, whereas Mm‐P cells could grow in the liquid medium but not in soft agar medium without ascitic fluid, and Mm‐S1 and Mm‐S2 cells could not grow in either medium. The ratio of the nuclear area to the cell area (NCR) of Mm‐A cells was a high (51%) but those of Mm‐Sl and Mm‐S2 cells were low (40–41%), and that of Mm‐P cells was intermediate (44%). The leukemogenicity of Mm‐1 cell lines was roughly correlated with their NCR. The possibility that interactions between Mm‐1 variant cells and host immune cells might be involved in the mechanisms of their different leukemogenicities was not supported by results on the in vitro susceptibilities of Mm‐1 variant cells to the cytostatic actions by normal macrophages and spleen cells and on leukemogenicities of the Mm‐1 variant cells in athymic nude mice. A possible method of control of the leukemogenicity of Mm‐1 variant cells is discussed.
Cultured mouse marrow cell lines: Interactions between fibroblastoid cells and monocytes
Authors: Zipori Dov , Friedman Aharon , Tamir Merana , Silverberg David , Malik Zvi
Published: February 2005

Continuous cell lines were derived from primary cultures of adherent bone marrow cells from SJL/J, BALB/c, C3H/eb, RF, and nude‐ICR mice. All these lines readily assumed a pure fibroblastoid appearance with the exception of the BALB/c line (MBA‐14), which retained both fibroblastoid and monocytoid cells. This particular line could promote the proliferation of myeloid progenitors (CFU‐C) in short‐term bone‐marrow cultures. The two cell types that composed the MBA‐14 cell line were successfully isolated and grown separately; the monocytes as the 14M and 14M1 cell lines and the fibroblastoid cells as the 14F clones. The latter were found to be preadipocytes and accumulated fat in the absence of added hydrocortisone, in medium supplemented with fetal calf serum. Growth of the monocyte lines (14M and 14M1) was dependent upon the mononuclear phagocyte stimulator CSF‐1. In the parent MBA‐14 cell line the growth of monocytes seemed to depend upon stimulating factor(s) produced by the fibroblastoid cells. The 14M1 monocytes were able to process and degrade antigen as efficiently as primary macrophages. Furthermore, processed antigen produced by 14M1 cells evoked proliferative response by antigen‐primed lymph‐node cells. In addition to these immunological functions the 14M1 cells were capable of modulating the colony‐stimulating activity and degree of adipogenesis exhibited by the fibroblastoid cells. These interactions between monocytes and fibroblastoid cells may constitute part of the mechanism controlling the activity of the hematopoietic microenvironment.
Dependence of HL‐60 myeloid cell differentiation on continuous and split retinoic acid exposures: Precommitment memory associated with altered nuclear structure
Authors: Yen Andrew , Reece Sara L. , Albright Kevin L.
Published: February 2005

The cell differentiation of HL‐60 human leukemic promyelocytes along the myeloid pathway due to various continuous and distributed exposures to retinoic acid was studied. HL‐60 myeloid differentiation was a continuously driven process; significant terminal cell differentiation occurred only after a minimum exposure to inducer of two division cycles. Cells so committed to differentiation retained a heritable, finite memory of differentiation commitment over a further division cycle. Prior to becoming committed, cells acquired precommitment memory of exposure to inducer. Precommitment memory abbreviated the subsequent exposure to inducer needed for commitment to differentiation. Precommitment memory was semistable. It was heritable, but was lost after four division cycles. The acquisition and loss of precommitment memory correlated with alterations in nuclear architecture detected by narrow angle light scatter using flow cytometry. The altered nuclear architecture first occurred before any overt cell differentiation or growth arrest. It was thus an early event in the induced program of terminal cell differentiation. Alterations in relative abundances of cytoplasmic proteins also occurred prior to overt cell differentiation or growth arrest. One of these was a 17 kdalton, anionic, probably Ca2+ binding, protein. Retinoic acid thus induced early cellular changes, including cytoplasmic and nuclear alterations, within one cell cycle when cell differentiation was not yet apparent.
Anion requirement and effect of anion transport inhibitors on the response of vero cells to diphtheria toxin and modeccin
Authors: Sandvig Kirsten , Olsnes Sjur
Published: February 2005

The anion requirement for toxic action of diphtheria toxin and modeccin was studied. In Cl−‐free Hepes buffer made isotonic with mannitol, cells were insensitive to diphtheria toxin and modeccin. Just 2 mM NaCl was sufficient to obtain full toxic activity of modeccin, whereas 140 mM NaCl was required for maximal intoxication with diphtheria toxin. Br− could substitute for Cl−. NO3−, I− and ClO3− were less efficient than Cl−, whereas SO42− and SCN− were unable to replace Cl−. Cl− deprivation both reduced the ability of cells to bind diphtheria toxin and prevented bound toxin from intoxicating the cells. The binding of modeccin was not reduced. SITS (4‐acetamide‐4′‐isothio‐cyano‐stilbene‐2,2′‐disulfonic acid), an inhibitor of Cl− entry, protected against diphtheria toxin and modeccin, indicating that Cl− transport is required for intoxication.
An increase in adenylate cyclase activity precedes DNA synthesis in cultured vascular smooth muscle cells
Authors: Franks D. J. , Plamondon J. , Hamet P.
Published: February 2005

Adenylate cyclase activity in cultured rat aortic vascular smooth muscle cells showed a linear correlation with the rate of DNA synthesis. When smooth muscle cells were rendered quiescent by shifting them from a serum‐supplemented medium to a medium containing low concentrations of plasma, the cells could be stimulated to proliferate by the addition of serum or by addition of a crude preparation of platelet‐derived growth factor. DNA synthesis began at 16 hours and was maximal at 24 hours. Prior to synthesis of DNA there was an increase in adenylate cyclase activity with a peak at 12 hours. Adenylate cyclase activity returned to basal level before DNA synthesis began. The increase in adenylate cyclase activity was not blocked by cycloheximide. Adenylate cycase activity could also be increased by incubating vascular smooth muscle cells with cholera toxin; however, the time course and magnitude of this increase was different from that caused by growth stimulants. Cholera toxin caused a slight increase in DNA synthesis at 16 hours, but was also cytotoxic to smooth muscle cells. An increase in adenylate cyclase activity may be a prerequisite for the progression from G1 to S.
Regulation of pyruvate kinase expression and growth in P‐815 mastocytoma cells. II. Potential role of prostaglandins
Authors: Ngo Julie‐Lani , Orlando Robert A. , Ibsen Kenneth H.
Published: February 2005

P‐815 mastocytoma cells increase the level of pyruvate kinase (PK) expression in response to chloroform‐methanol extracts of conditioned media, butyrate, and dibutyryl cyclic AMP ( but2cAMP) plus theophylline. The butyrate effect is indomethacin sensitive, suggesting a prstaglandin (PG) is the active signaling factor. Moreover, the chloroform‐methanol extracts contain PGE2 and PGF2α and additions of the latter enhance PK activity. PGE2α alone has little or no effect but acts synergistically with PGF2. These data show that PGF2α can regulate PK levels. On the other hand, other factors may also be active, since the endogeneous and the but2cAMP plus theophylline effects are indomethacin insensitive. Most of the factors that increase PK activity also inhibit cellular growth; however, regulation of PK expression can be uncoupled from growth inhibition.
Transaminase inhibitors block glycolysis and G1 to S phase progression in chick embryo fibroblasts: Reversal by α‐keto acids
Authors: Groelke John , Amos Harold
Published: February 2005

Two transaminase inhibitors, aminooxyacetate and cycloserine, inhibited the initiation of insulin‐stimulated DNA synthesis in chick embryo fibroblasts. This inhibition was overcome when pyruvate (4 mM), oxaloacetate (4 mM), or α‐ketobutyrate (10 mM) was included in the culture medium with hormone and inhibitor. Aminooxyacetate also inhibited lactate production in insulin treated cultures in the absence of added α‐keto acid.
Biological properties of a hepatocyte growth factor from rat platelets
Authors: Russell William E. , McGowan Joan A. , Bucher Nancy L. R.
Published: February 2005

In an accompanying communication we demonstrated that about half of the potency of rat serum to stimulate DNA synthesis in cultured adult rat hepatocytes resides in a polypetidelike substance from the platelets. A lysate of rat platelets was able to restore the potency of platelet‐poor rat serum, whereas a lysate of human platelets inhibited thymidine incorporation by the hepatocytes. Moreover, addition to these cultures of either highly purified human platelet‐derived growth factor (PDGF) or human platelet factor 4 (PF‐4) failed to influence DNA synthesis either alone or in the presence of rat or human platelet‐poor serum, which is required for expression of PDGF activity. Unlike the human platelet factors, rat platelet lysate (RPL) was moderately active by itself and was augmented equally well by platelet‐poor serum from either source. At concentrations below 5%, platelet‐poor serum from hypophysectomized rats was as potent as that from normal rats in augmenting RPL activity. This suggests that, unlike PDGF, which is not activated by hypophysectomized rat serum, the hepatotrophic component of RPL does not require the presence of exogenous somatomedins for activity, but interacts instead with other plasma constituents or with somatomedins produced by the hepatocytes in vitro. Rat platelets do, however, appear to contain PDGF or its rat equivalent in addition to the hepatocyte growth factor, since if they are heated to 100°C for 10 min, their ability to stimulate nuclear labeling in confluent BALB/c 3T3 cells is not impaired, while their ability to stimulate DNA synthesis in rat hepatocytes is destroyed. These studies indicate that the hepatocyte growth factor from rat platelets differs from PDGF in its biological as well as physical characteristics, but that rat platelets also contain PDGF or an equivalent substance.
Maintenance of granulocyte‐monocyte progenitor cells in liquid cultures of human foetal liver
Authors: Toksoz Deniz , Brown Geoffrey
Published: February 2005

These studies describe a liquid suspension culture system for normal myeloid cells derived from human foetal liver. A simple one‐step fractionation procedure was employed to obtain a cell population capable of expanding into all stages of myeloid differentiation, including committed myeloid progenitor cells (GM‐CFC). Cell proliferation in these cultures resulted in the maintenance of early myeloid populations for up to a month. In order to extend myeloid cell maintenance, a specific factor in the form of media conditioned by human endothelial cells (endo C.M.) was used. Addition of endo C.M. to foetal liver cultures resulted in increased myeloid proliferation coupled to extensive myeloid differentiation. Clonally derived foetal liver culture cells proliferated for up to 2 months in the presence of endo C.M. before maturing into macrophages. These results show that endo C.M. exert an extensive proliferative effect on early myeloid cells as well as inducing their differentiation. The large quantity of cells in early stages of myeloid differentiation provided by foetal liver cultures may be useful for biochemical and molecular biology studies of myelopoiesis. In addition, these cultures are a potential source from which to derive normal myeloid lines. The separation of the potent proliferative activity present in endo C.M. may yield an effector which maintains human myeloid cell proliferation.
Insulin inhibits the glucocorticoid‐mediated increase in hepatocyte EGF binding
Authors: Lin Qixiong , Blaisdell Joyce , O'Keefe Edward , Earp H. Shelton
Published: February 2005

Hydrocortisone and dexamethasone produced a time‐dependent increase [125I]epidermal growth factor ([125I]EGF) binding in primary cultures of isolated rat hepatocytes. Maximally effective doses of glucocorticoids resulted in a 70–100% increase in binding. The effect was similar when hepatocytes were maintained on collagen‐coated plates or directly on culture dishes. The glucocorticoid‐mediated increase in [125I]EGF binding could be detected after 4 h exposure to glucocorticoid and was substantial by 8 h. The major effect of glucocorticoid appeared to be to increase the number of EGF receptors. While insulin (100 nM) had no effect on basal [125I]EGF binding, it significantly inhibited the increase produced by the glucocorticoid. Since the inhibitory effect of insulin was only observed when insulin was added with the inducing glucocorticoid, insulin appears to inhibit an early hydrocortisone‐mediated event.
Rb+ influx in response to changes in energy generation: Effect of the regulation of the ATP content of HELa cells
Authors: Ikehara Toshitaka , Yamaguchi Hisao , Hosokawa Keiko , Sakai Tetsuhiro , Miyamoto Hiroshi
Published: February 2005

The effect of changes in energy metabolism on Rb+ influx was studied in HeLa cells. Irrespective of whether ATP production was controlled by varying the activity of glycolysis or of oxidative metabolism on addition of certain combinations of glucose, carbonylcyanide m‐chlorophenylhydrazone, monoiodoacetic acid, and quercetin, Rb+ influx changed as a linear function of the ATP content, which varied in a wide range up to the normal level (15–20 nmol/mg protein or 3–4 mM). The difference between results obtained by these procedures was not significant. As the intracellular Na+ content varied at different ATP contents, the Na+ content was adjusted to similar levels by chilling the cells with varying ATP contents. However, a linear relation was still observed. A similar dependence was also obtained for cytoplasmic ATP, which would be more closely connected with the Na, K‐pump than total ATP. The ratio of ouabain‐sensitive Rb+ influx to the corresponding part of lactate production was nearly 2 in the presence of 2 mM glucose. From these results it is concluded that (1) active Rb+ influx, which is chiefly maintained by energy generated through glycolysis, can also be supported by oxidative metabolism; (2) Rb+ influx is regulated linearly as a function of the cellular ATP content up to the control level; but does not increase if ATP is raised still further; and (3) 2 Rb+ ions move concomitantly at the expense of one ATP molecule.
Analysis of cell division by time‐lapse cinematographic studies of hydrocortisone‐treated embryonic lung fibroblasts
Authors: Absher Marlene , Cristofalo Vincent J.
Published: February 2005

Hydrocortisone is a modulator of cell division and has been shown to prolong the replicative in vitro life span of human embryonic lung fibroblasts. Time lapse cinematography was used to analyze the proliferative behavior of individual cells in populations of fibroblasts exposed to hydrocortisone in young cultures during a single growth cycle and in aged cultures that had been continuousiy exposed to hydrocortisone. Results indicate that hydrocortisone causes a decrease in the interdivision time (IDT) of a portion of the cells in the population and this effect is augmented after continuous exposure to hydrocortisone. Hydrocortisone does not appear to increase the number of initial dividers in the population but increases growth rate in the early stages of the culture period. Analysis of mother‐daughter IDT pairs further suggests that hydrocortisone exerts its effects on IDT independently for a given cell.
Mitogenic and comitogenic properties of the indole alkaloid class of tumor promoters
Authors: Novogrodsky Abraham , Patya Miriam , Fujiki Hirota , Rubin Albert L. , Stenzel Kurt H.
Published: February 2005

We determined the mitogenic and comitogenic properties of tumor promoters in the indole alkaloid series; agents that differ structurally from 12–0‐tetradecanoylphorbol‐13‐acetate (TPA), which is known to be a lymphocyte mitogen. Teleocidin and dihydroteleocidin B were mitogenic for human lymphocytes, and lyngbyatoxin A elicited little or no mitogenesis. Catalase enhanced the mitogenicity of teleocidin and dihydroteleocidin B, and lyngbyatoxin A was also mitogenic in the presence of catalase. The potency of the agents was TPA = teleocidin > dihydroteleocidin B > lyngbyatoxin A. Dimethylsulfoxide and butyric acid markedly inhibited proliferation induced by these agents in human lymphocytes. The indole alkaloid tumor promoters were all comitogenic for murine thymocytes and induced production of interleukin‐2. While the comitogenic effect of teleocidin was similar to that of TPA in its susceptibility to inhibition by dimethylsulfoxide and butyric acid, the comitogenic effect of dihydroteleocidin B and lyngbyatoxin A were less susceptible to these agents. These findings may facilitate the identification of cellular sites responsible for the inhibitory effect of differentiating agents on proliferative responses of lymphocytes.
Effect of retinoic acid on the proliferation and phagocytic capability of murine macrophage‐like cell lines
Authors: Goldman Rachel
Published: February 2005

Retinoic acid (RA) exerted a variable degree of growth inhibitory activity on the macrophage‐like cell lines P388D1, J774.2, WEHI‐265, WEHI‐3, and PU‐5. Comparison of cell proliferation and clonal growth suggests that at concentrations of 10−9‐10−6 M the inhibitory activity stems from processes leading to elongation of cell cycle time and not from terminal differentiation processes. RA was shown to be a potent inducer of the development of high‐phagocytic phenotypes (assessed by phagocytosis of heat‐killed yeast cells) in the P388D1, J774.2, and WEHI‐265 cell lines which differ substantially in their proliferative and adherence characteristics. The PU‐5 and WEHI‐3 cell lines were not induced by RA to express an enhanced phagocytic activity toward heat‐killed yeast cells. The augmented phagocytic capability was dose dependent over a wide range of RA concentrations. In P388D1 cells, 2 × 10−12 M RA already exerted significant phagocytosis augmentation effects, which progressively increased up to 2 × 10−5 M RA, the highest concentration tested. Retinal, retinyl acetate, and retinol had similar effects to those of RA on both cell adherence and phagocytosis in P388D1 cells, albeit at concentrations four to six orders of magnitude higher. Optimal development of the high‐phagocytic phenotype in P388D1, J774.2, and WEHI‐265 cells required at least 96 hr of culture in the presence of RA; at 48 hr and 23 hr the effects were already substantial, whereas at 4 hr of exposure to RA no significant enhancement of phagocytosis could be detected. Thus both extended periods of culture in the presence of RA (more than two to three cell cycles) and high concentrations were needed for induction, in more than 90% of the cells, of the expression of a high‐phagocytic phenotype. The reversion to a low‐phagocytic phenotype upon removal of RA was also rather slow and required several cell cycles. In P388D1 cells RA also enhanced the phagocytosis of latex beads but had no effect on the phagocytosis of starch particles, or the extent of binding of immunoglobulin G‐coated sheep red blood cells (SRBC). The expression of receptors for concanavalin A and for nonopsonized SRBC remarkably increased in RA‐treated cells, as was the ability to perform Fc‐receptor mediated erythrophagocytosis. Both P388D1 cells and WEHI‐265 cells were induced by RA to express nitroblue tetrazolium reducing activity. The data suggest that RA induces profound changes in the functional capabilities of macrophage‐like cell lines which are apparently not dependent on cessation of growth and terminal differentiation processes.
Mutants of chinese hamster cells deficient in thymidylate synthetase
Authors: Li I‐Chian , Chu Ernest H. Y.
Published: February 2005

Stable mutants of Chinese hamster V79 cells deficient in thymidylate synthetase (TS; E.C. 2.1.1.45) have been selected from cultures grown in medium supplemented with folinic acid, aminopterin, and thymidine (FAT). After chemical mutagenesis, the frequency of colonies resistant to the “FAT” medium increased more than 100‐fold over the spontaneous frequency. The optimal expression time of the mutant phenotype was 5–7 days after mutagen treatment. The recovery of FAT‐resistant colonies in the selective medium was not affected by the presence of wild‐type cells at a density below 9,000 cells per cm2. All 21 mutants tested exhibited thymidine auxotrophy; neither folinic acid nor deoxyuridine could support mutant cell growth. There was no detectable TS activity in all 11 mutants so far examined and only about 50% of wild‐type activity in three prototrophic revertants, as measured by whole‐cell and cell‐free enzyme assays. The apparent Michaelis‐Menten constant (Km) for deoxyuridine‐5′‐monophosphate and inhibition constant (Ki) for 5‐fluorodeoxyuridine‐5′‐monophosphate, measured by whole‐cell enzyme assay, appear to be similar for the wild‐type and revertant cell lines. Using 5‐fluoro‐[63H]‐2′‐deoxyuridine 5′‐monophosphate as active site titrant, the relative amounts of TS in crude cell extract from the parental, revertant, and mutant cells were shown to exist in a 1:0.5:0 ratio. Furthermore, the enzymes from two revertants were more heat labile than that of V79 cells. These properties, taken together, suggest that the FAT‐resistant, thymidine auxotrophic phenotype may be the result of a structural gene mutation at the TS locus. The availability of such a mutant facilitates studies on thymidylate stress in relation to DNA metabolism, cell growth, and mutagenesis.
Endocytosis and exocytosis of protein in capillary endothelium
Authors: Williams Stuart K. , Greener Deborah A. , Solenski Nina J.
Published: February 2005

The transport of proteins across continuous capillary endothelium is believed to be mediated by micropinocytic vesicles which shuttle molecules between the lumenal and abluminal plasma membrane. We have studied the ability of capillary endothelial cells isolated from rat epididymal fat to endocytose fluorescently labelled ovalbumin within micropinocytic vesicles. Net association of fluorescent ovalbumin with endothelial cells reaches an equilibrium after 40 minutes of incubation. This equilibrium is presumably due to a balance between endocytosis and subsequent exocytosis of this protein. Capillaries equilibrated with fluorescent ovalbumin exhibited rapid exocytosis of this protein when it was removed from the external medium. The rate of endocytosis was concentration dependent and obeyed the kinetics expected for adsorptive phase endocytosis. High concentrations of ovalbumin stimulated the ingestion of 14C‐sucrose, a marker of fluid endocytosis, suggesting that protein can affect the movement of vesicles within the endothelial cytoplasm. These results imply that capillary endothelium isolated from rat epididymal fat exhibits the ability to endocytose and subsequently exocytose protein. This demonstrates that the two components of endothelial vesicular transport or transcytosis can be observed and studied in a system of isolated capillary endothelium.
Dependence of lethality induced by a direct DNA perturbation of synchronized human diploid fibroblasts on different periods of the DNA synthetic period (S Phase)
Authors: Tsutsui Takeki , Ohmori Manabu , Suzuki Nobuko , Maizumi Heiji
Published: February 2005

The cytotoxic effect of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of cultured human diploid fibroblasts was examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2‐hour periods during the S phase with 5‐bromodeoxyuridine (0.1–10 μM), followed by irradiation with near‐UV (5–10 min). The 5‐bromodeoxyuridine‐plus‐irradiation treatment was cytotoxic, while treatment with 5‐bromodeoxyuridine alone or irradiation alone was not cytotoxic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 hours of S phase, whereas scarce lethality was observed in late S phase. The extent of substitution of 5‐bromodeoxyuridine for thymidine in newly synthesized DNA was similar in every period of the S phase. Furthermore, no specific period during S phase was significantly more sensitive to treatment with respect to DNA damage, as determined by an induction of unscheduled DNA synthesis. These results suggest that a certain region or regions in the DNA of human diploid fibroblasts, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by 5‐bromodeoxyuridine treatment plus near‐UV irradiation for cell survival.
The role of polyamines in somatomedin‐stimulated differentiation of L6 myoblasts
Authors: Ewton Daina Z. , Erwin Bradley G. , Pegg Anthony E. , Florini James R.
Published: February 2005

The somatomedins are potent stimulators of proliferation and differentiation of cultured myoblasts. In studies on the mechanism(s) of these actions, we have measured the activities of ornithine decarboxylase (ODC), an enzyme associated with rapid cell proliferation, and creatine kinase (CK), a biochemical marker for muscle differentiation, after treatment of L6 myoblast cultures with Multiplication Stimulating Activity (MSA), a member of the somatomedin family of insulinlike growth factors. ODC levels reached a peak 24 hours after MSA addition (before any detectable differentiation of the myoblasts) and then decreased as differentiation commenced and CK activity increased. Addition of alpha‐difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, caused a dramatic decrease in differentiation. Measurement of 3H‐thymidine incorporation, DNA content, and cell number established that the effect of DFMO on differentiation was not a simple consequence of its antiproliferative actions. Cellular levels of putrescine and spermidine (but not spermine) decreased substantially following addition of DFMO to the cultures. The inhibitory effects of DFMO were abolished upon addition of exogenous polyamines to the medium. However, addition of polyamines in the absence of MSA or DFMO did not mimic the stimulation of differentiation by MSA. We conclude that polyamines play an essential role in the stimulation of L6 myoblast differentiation by somatomedins, but they are not sufficient to effect this stimulation.
Verapamil enhances the toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin and antitransferrin receptor with pseudomonas exotoxin
Authors: Akiyama Shin‐Ichi , Gottesman Michael M. , Hanover John A. , Fitzgerald David J. P. , Willingham Mark C. , Pastan Ira
Published: February 2005

Verapamil, a clinically important calcium channel blocker, has been found to cause a 40‐fold enhancement of killing of the human KB cell line by a cytotoxic conjugate of epidermal growth factor with Pseudomonas exotoxin (EGF‐PE). Synergistic effects of verapamil and EGF‐PE are also seen on HeLa D98 cells and a human epidermal carcinoma cell line, A431. Verapamil also potentiates the effect of a toxic conjugate formed between Pseudomonas exotoxin and a monoclonal antibody to the human transferrin receptor (anti‐TFR‐PE) and enhances the effect of Pseudomonas exotoxin (PE) alone. Two other calcium antagonists were tested. Diltiazem enhances the cytotoxic effect of EGF‐PE, but nifedipine does not. Verapamil does not affect the binding and uptake of 125I‐EGF by KB cells, but it significantly delays the disappearance of internalized 125I‐EGF from the cells. Density gradient fractionation studies using cell homogenates suggest that 125I‐EGF accumulates in an undegraded form in lysosomes when cells are treated with verapamil. By immunofluorescence microscopy using an antibody to PE, EGF‐PE was found to accumulate in lysosomes; by electron microscopy the lysosomes had an abnormal appearance. The effects of verapamil on toxicity of EGF‐PE and lysosomal function appear to be related. However, it is not known whether the enhanced toxicity of EGF‐PE in the presence of verapamil is due to its delayed degradation in lysosomes or some more general effect of verapamil on membrane permeability.
Characterization of the metabolic turnover of epidermal growth factor receptor protein in A‐431 cells
Authors: Stoscheck Christa M. , Carpenter Graham
Published: February 2005

The metabolism of the receptor for epidermal growth factor (EGF) in A‐431 cells has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti‐EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. The rate of EGF receptor degradation (t½12 = 20 hr) was faster than the rate of degradation of total cell protein (t½12 = 52 hr). When EGF was added at the beginning of the chase, the half‐life of prelabeled receptor decreased to 8.9 hr. This decrease was specific, as the level of total cellular protein and another plasma membrane protein, the transferrin receptor, were relatively unaffected by EGF. The carbohydrate portion of the receptor is degraded, in the presence or absence of EGF, at approximately the same rate as the protein moiety. The amount of EGF receptor protein in A‐431 cells has been quantitated by radiolabeling total cellular protein and quantitating the immunoprecipitable receptor. The EGF receptor constitutes approximately 0.15% of the total cell protein in A‐431 cells. These cells, therefore, have approximately 30 times more EGF receptor protein than fibroblasts. The EGF receptor constitutes an even higher proportion of 3H‐glucosamine‐ or 3H‐mannose‐labeled macromolecules in A‐431 cells, 1.5% or 5.2%, respectively. The EGF receptor from A‐431 cells can easily be identified by submitting carbohydrate‐labeled, solubilized cells to electrophoresis as described by Laemmli (1970).