bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin and growth factors stimulate rapid posttranslational changes in glucose transport in ovarian granulosa cells
Authors: Allen W. Ross , Nilsen‐Hamilton Marit , Hamilton Richard T.
Published: February 2005

The glucose analogues, 3‐0‐methyl‐D‐glucose and 2‐deoxy‐D‐glucose, have been used to characterize glucose transport and its regulation by serum and growth factors in monolayer cultures of granulosa cells obtained from bovine ovaries. Uptake of 3‐0‐methylglucose was shown to be independent of the Na+‐gradient, independent of energy, did not show accelerated exchange, and was stereospecific. Serum withdrawal resulted in a biphasic decrease in initial rates of glucose uptake with half‐times for the two phases of 50 minutes and 3 hours. Insulin could prevent the decrease in uptake rates with a half‐maximum concentration of 10.5 1/8 3 nM. Insulin was shown to stimulate DNA synthesis with a concentration of half‐maximum response of 28 nM.

Insulin or serum stimulation of 3‐0‐methylglucose uptake in serum‐starved cells resulted in a two to threefold increase in initial rates, with a time for halfmaximum stimulation of 3 minutes. The insulin‐stimulated increase was insensitive to cycloheximide and cyanide during the first 30 minutes, and this early, rapid stimulation was also produced by brain FGF (fibroblast growth factor), pituitary FGF, epidermal growth factor, calf serum, and some but not all samples of follicular fluid. Insulin also stimulated 2‐deoxyglucose and a‐aminoisobutyric acid uptake during the first 5 minutes of addition, and these early stimulations were shown to be posttranslational changes.
Growth inhibitory protein(s) in the 3T3 cell plasma membrane. Partial purification and dissociation of growth inhibitory events from inhibition of amino acid transport
Authors: Raben Dan , Lieberman Michael A. , Glaser Luis
Published: February 2005

It has been reported previously that a plasma membrane‐enriched fraction from 3T3 cells arrests the growth of sparse 3T3 cells early in the G1 phase of growth (Whittenberger et al., ′78, ′79). Addition of membranes to sparse 3T3 cells also results in a decrease of the rate of transport of αaminoisobutyric acid (Lieberman et al., ′79). We have partially purified the growth‐inhibitory proteins from plasma membrane of 3T3 cells. These growth‐inhibitory proteins behave as intrinsic membrane proteins and do not require membrane lipids for activity. The partially purified proteins arrest cell growth without affecting the rate of α‐aminoisobutyric transport; thus, inhibition of both transport and cell growth are not obligatorily coupled events.
Rates of incorporation of radioactive molecules during the cell cycle
Authors: Gray J. W. , Pallavicini M. G. , George Y. S. , Groppi V. , Look M. , Dean P. N.
Published: February 2005

We report measurements of the incorporation of radioactive molecules during short labeling periods, as a function of cell‐cycle stage, using a cell‐sorter‐based technique that does not require cell synchronization. We have determined: (1) tritiated thymidine (3H‐TdR) incorporation throughout S‐phase in Lewis lung tumor cells in vitro both before and after treatment with cytosine arabinoside; (2) 3H‐TdR incorporation throughout S‐phase in KHT tumor cells in vitro and in vivo; (3) 3H‐TdR incorporation throughout S‐phase in Chinese hamster ovary cells and compared it with DNA synthesis throughout S‐phase; (4) a mathematical expression describing 3H‐TdR incorporation throughout S‐phase in Chinese hamster M3‐1 cells; and (5) the simultaneous incorporation of 3H‐TdR and 35S‐methionine as they are related to cell size and DNA content in S49 mouse lymphoma cells. In asynchronously growing cells in vitro and in vivo, 3HH‐TdR incorporation was generally low in early and late S‐phase and highest in mid‐S‐phase. However, in Lewis lung tumor cells treated with cytosine arabinoside 3H‐TdR incorporation was highest in early and late S‐phase and lowest in mid‐S‐phase. Incorporation of 35S‐methionine increased continuously with cell size and DNA content. Incorporation of 3H‐TdR in CHO cells was proportional to DNA synthesis.
Trifluoperazine inhibits spreading and migration of cells in culture
Authors: Connor Charles G. , Brady Richard C. , Brownstein Barbara L.
Published: February 2005

Trifluoperazine (TFP) blocks spreading and migration of cultured mammalian cells. These are calcium‐dependent and microfilament‐mediated processes. Calmodulin, a regulator of many calcium‐dependent processes in cells, is selectively inhibited by TFP. Cell spreading on a plastic‐ or collagen‐coated substratum was reversibly inhibited by 10 μM TFP. The drug blocks cell spreading even in the presence of 1 mM cAMP. TFP is as effective as cytochalasin B (CB), an inhibitor of microfilament function, in blocking cell spreading. All cell lines tested, whether “normal” or virally transformed, failed to spread in TFP. The drug, at a concentration sufficient to inhibit spreading, does not interfere with the initial attachment of a cell to a plastic surface. Cells plated in the presence of 10 μM TFP attach at a rate and to an extent equal to untreated controls. TFP added to already spread cells results in a reversible cell rounding. Detection of fibronectin by indirect immunofluorescence suggests TFP‐induced cell rounding is not due to shedding of fibronectin from the cell surface. TFP reversibly blocks cell migration into a wound edge almost as effectively as CB. We suggest that TFP interferes with these microfilament‐mediated functions by direct action on the microfilaments or indirect action by inactivating calmodulin.
Expression of angiotensin‐converting enzyme activity in cultured pulmonary artery endothelial cells
Authors: del Vecchio Peter J. , Smith James R.
Published: February 2005

Angiotensin‐converting enzyme (EC 3.4.15.1) is a carboxyterminal dipeptidyl peptidase. The enzyme catalyzes the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II. In addition, the enzyme catabolizes bradykinin. Because of these actions, the enzyme is of pivotal importance in blood pressure homeostasis. Numerous investigators have demonstrated the presence of the enzyme in association with endothelial cells but relatively little is known concerning the factors controlling the expression of enzyme activity by endothelial cells in culture. We have demonstrated that endothelial cells in culture do not express significant amounts of enzyme activity until several days after growth ceases due to high cell density. This is important because it demonstrates a change in function with stage of growth in culture and a possible difference in functional capabilities between nondividing endothelial cells and cells that are dividing in response to injury. Since density‐dependent expression of differentiated traits does not appear to be unique to endothelial cells an understanding of the mechanisms underlying this phenomenon may provide a general explanation for the expression of differentiated traits by cultured cells.
Precursor of mast cells fixed in the skin of mice
Authors: Matsuda Hiroshi , Kitamura Yukihiko , Sonoda Takashi , Imori Tatsuo
Published: February 2005

The presence and origin of mast‐cell precursors fixed in the skin tissue of mice were investigated. Giant granules of beige (C57BL/6‐bgj/bgj, Chediak‐Higashi syndrome) mice were used to distinguish different populations of mast cells. Pieces of the skin were grafted from the intact WBB6F1 (WB × C57BL/6)F1−+/+ mice onto the back of the WBB6F1−+/+ mice which had been irradiated and injected with bone marrow cells of C57BL/6‐bgj/bjj mice (bgj/bgj −+/+ chimeras). Although the number of mast cells in the skin grafts decreased after the transplantation, the mast‐cell precursors circulating in the bloodstream of bgj/bgj −+/+ chimeras (bgj/bgj type) did not seem to enter into the skin grafts, because most of mast cells were of +/+ type after the recovery of mast‐cell number to pregrafting levels. As a considerable proportion of +/+‐type mast cells was labeled with 3H‐thymidine, the recovery of mast‐cell number in the grafts was attributed to the proliferation and differentiation of +/+‐type precursor cells fixed in the skin tissue of the donor. On the other hand, the skin of WBB6F1‐W/Wv mice seemed to be depleted of fixed precursors, because most of mast cells were of bgj/bgj type in skin grafted from WBB6F1‐W/Wv mice to bgj/bgj−+/+ chimeras. Since the fixed precursor cells which proliferate and differentiate into mast cells after skin grafting may be transferred to WBB6F1‐W/Wv mice by bone marrow transplantation, such precursor cells seem to have been derived from the bone marrow.
Identification of the glucose/glycosylation‐regulated proteins as those which accumulate in the temperature‐sensitive cell line K12
Authors: Melero José A.
Published: February 2005

The analysis of proteins synthesized by the temperature‐sensitive cell line K12 at the restrictive temperature or after glucose starvation revealed that two main polypeptides were enhanced under both conditions. Besides having the same apparent molecular weights, these new proteins showed identical patterns of partial proteolysis products, irrespective of being isolated from cells incubated at the nonpermissive temperature or from glucose‐starved cultures. These results identified the proteins overproduced by K12 cells at 39.5 °C as those described in other cell systems, called „glucose/glycosylation regulated proteins”︁ (GR proteins). In addition, it is shown that K12 cells are defective in some step of the glycosylation pathway; this defect may be responsible for the overproduction of GR proteins in K12.
Effect of substrata and fibroblast growth factor on the proliferation in vitro of bovine aortic endothelial cells
Authors: Gospodarowicz D. , Lui G.‐M.
Published: February 2005

The hypothesis that, in the case of clonal or low‐density cultures, cells which do not readily proliferate are those that do not produce an extracellular matrix (ECM), while those that proliferate actively are cells that have retained their ability to produce it, has been tested using low‐density vascular endothelial cell cultures maintained on either plastic or ECM‐coated dishes and exposed to various combinations of media and sera.

Proliferation of low‐density vascular endothelial cell cultures seeded on plastic and exposed to DMEM, RPMI‐1640, or medium 199 plus thymidine is a function of the batch of calf serum used to supplement the various media. In all three cases, such cultures proliferated at a slow rate and fibroblast growth factor (FGF) greatly accelerated their proliferation. In contrast, when similar cultures were seeded on ECM‐coated dishes, they actively proliferated regardless of the batch of calf serum to which they were exposed. FGF was no longer required in order for cultures to be come confluent. In the case of cultures exposed to RPMI‐1640 or medium 199 plus thymidine, it was even toxic.

When cultures were exposed to either medium 199 or Waymouth medium, cells did not proliferate, regardless of the substrate (either plastic or ECM) upon which they were maintained and of the batch of serum to which they were exposed. Addition of FGF to such media had no effect. It is therefore likely that nutrient limitations in both of these media restrict the ability of low‐density vascular endothelial cells to respond to the mitogenic stimuli provided by either serum of FGF. These restrictions cannot be relieved by maintaining cells on ECM‐coated dishes, and modifications of the nutrient composition of both media is required in order to allow cells to respond to either FGF or serum when maintained on plastic or to serum alone when maintained on ECM.

These results suggest that, when low‐density cell cultures are maintained on plastic and exposed to an adequate medium, their proliferation will be a function of both serum and FGF. When maintained on ECM, their proliferation will depend only on serum. It is therefore possible that the inability of serum to stimulate optimal cell proliferation when cells are maintained on plastic results from an inability of the cells to produce an ECM, and that FGF could induce such production.
Patterns of cell communication and differentiation in SV40 transformed human keratinocytes
Authors: Steinberg Mark , Defendi Vittorio
Published: February 2005

Fluorescein dye microinjection was used to demonstrate changes in communication between human epidermal keratinocytes grown in vitro after infection by the oncogenic virus, SV40. Whereas keratinocytes are normally fully coupled to each other, dye spread becomes progressively restricted to small cell subpopulations after infection, although dye coupling is increased when the infected cells attain high densities. Reduction or enhancement of dye coupling is correlated with similar changes in the extent of cytochemical differentiation and cornified envelope formation.
The synthesis of protein(S) for chromosome condensation may be regulated by a post‐transcriptional mechanism
Authors: Nishimoto Takeharu , Ishida Ryoji , Ajiro Kozo , Yamamoto Susumu , Takahashi Taijo
Published: February 2005

A temperature sensitive mutant of BHK21, tsBN2, showed a premature chromosome condensation (PCC) upon the temperature shift of 40.5°, even in the absence of DNA replication. The induction of PCC requires new protein synthesis, but not necessarily new RNA synthesis. Our data suggested that the messenger RNA for chromosome condensation starts to be transcribed at the beginning of Sphase. At the permissive temperature (33.5°), the messenger RNA for chromosome condensation translated with a very slow rate during S phase and rapidly in G2‐M phase. At the nonpermissive temperature (40.5°), however, those messenger RNAs were translated anytime, so that various figures of PCC appeared depending on the cell cycle. On the way of PCC induction, ribosomal RNA synthesis was inhibited at first, as expected from mitosis. Our data suggested that the synthesis of protein(s) for chromosome condensation was regulated by the post‐transcriptional mechanism, in which tsBN2 might be defective, especially at the translational level.
Pulmonary macrophages alter the collagen phenotype of lung fibroblasts
Authors: Kelley Jason , Trombley Lucy , Kovacs Elizabeth J. , Davis Gerald S. , Absher Marlene
Published: February 2005

Cultured lung fibroblasts produced and secreted interstitial collagen types I and III. The relative proportion of type III collagen increased as a linear function of cell density, with confluent cultures producing 8.6% type III collagen. When human lung fibroblasts were cultured in the presence of newly harvested lung macrophages, the proportion of type III collagen secreted rose to 15.5%. This high level of type III collagen synthesis was greater than could be induced by withdrawal of serum, a perturbation known to alter the proportion of types I and III collagen synthesized by fibroblasts. This effect on fibroblast phenotype was independent of cell density, as both low and high density cultures of fibroblasts responded similarly when cultured with macrophages. There was no evidence that fibroblasts synthesize new or different collagen types (such as type I trimer) in response to macrophages. Optimal conditions for eliciting an effect on fibroblast connective tissue metabolism required interaction of the two cell types for 5–8 days. These in vitro changes are analogous to the sequence of interactions and changes in connective tissue metabolism seen during recovery from tissue injury.
In vitro differentiation of chicken embryo skin cells transformed by rous sarcoma virus
Authors: Yoshimura Mokoto , Kaji Akira , Iwasaki Yuzo
Published: February 2005

The epidermal cells isolated from 14‐day chicken embryo shank skin epidermis were infected in vitro with Rous sarcoma virus (RSV). Within a few weeks, rapidly growing colonies of epithelial cells appeared among the sea of transformed fibroblastic cells. When isolated and subcultured, these cells were found to possess typical markers of skin epidermis. The presence of major keratin and typical epithelial cell type morphology strongly suggested that these cells were transformed epidermal cells retaining their differentiated characteristics but having the capacity to propagate in cell culture. If RSV tsNY68, an RSV mutant having a temperature lesion in the src gene, was used, similar transformed epidermal cells were obtained at 36°C (permissive temperature). At the nonpermissive temperature (41°C) the growth rate of these cells decreased and additional keratin species appeared. At 41°C the cells were flattened and lost the refractivity in their peripheries. All the keratins which are synthesized at the nonpermissive temperature were present in normal differentiated shank skin of 19‐day old chick embryo. These cells also had “cornified envelop,” indicating extensive differentiation. Viral production was as efficient as transformed fibroblasts during the rapid growth phase, while it declined significantly after the cells reached confluency, exhibiting the differentiated characteristics. Since no normal epidermal cells could be cultured under our experimental conditions, these results represent examples in which the src gene is essential for propagation of differentiated cells in cell culture while it abolishes only a part of differentiated characteristics.
Post‐transcriptional control of protein synthesis in Balb/C‐3T3 cells by platelet‐derived growth factor and platelet‐poor plasma
Authors: Cochran Brent H. , Lillquist Jay S. , Stiles Charles D.
Published: February 2005

Platelet‐derived growth factor (PDGF) and platelet‐poor plasma, which lacks PDGF, both induce a rapid increase in the rate of total protein synthesis within quiescent, density‐arrested Balb/c‐3T3 cells. This stimulation of protein synthesis is associated with an increased aggregation of ribosomes into polyribosomes. Nuclear functions are not required for this response, as demonstrated by the observation that this stimulation of protein synthesis occurs in cells pretreated with actinomycin D and in enucleated cells (cytoplasts). The response to PDGF persists even after PDGF has been removed from the culture medium, but in contrast, when plasma is removed from the medium, polysomes dissaggregate and protein synthesis declines. PDGF and plasma do not function synergistically to increase protein synthesis, whereas they do to induce optimum DNA synthesis. Thus stimulation of the translational apparatus may be necessary for the mitogenic response of Balb/c‐3T3 cells to growth factors, but it is not by itself sufficient.
A molecular mechanism of regulation for granulocyte macrophage colony stimulating factor from T lymphocytes
Authors: Frölich Marianne
Published: February 2005

We have partially purified a mitogenic factor, termed colony stimulating factor, which is secreted from a human T lymphocytic cell line, and which stimulates in vitro formation of colonies of macrophages and granulocytes by human bone marrow cells. Colony stimulating factor (CSF) produced by cells grown in serum supplemented media has a molecular weight of 60,000. It easily dissociates into a low (20,000) and a high (40,000) molecular weight component. The low molecular weight component, F I, initiates colony formation by a subset of the marrow cells. F II, the high molecular weight component, is inactive by itself, but reconstitution of F I with F II causes a dramatic increase in the number of target cells that form colonies. In serum‐free media the T‐cell line secretes only F I, which can be activated by F II. F II is only present when serum has been added to the medium, yet it is not present in the serum. Therefore, either the lymphocytes modify a component of the serum or they are stimulated to secrete F II by the serum. The molecular properties of this CSF suggest a possible mechanism of regulation of similar factors in that dissociation and reassociation may be physiologically important in determining which cell population is activated in hematopoesis.
Large‐scale preparation and characterization of human colony‐stimulating factor
Authors: Okabe Tetsuro , Nomura Hitoshi , Sato Noriharu , Ohsawa Nakaaki
Published: February 2005

In order to obtain human granulocytic colony‐stimulating factor (G‐CSF) in large quantities, a large‐scale culture system of human G‐CSF‐producing cells has been established. The cell used for this system was T3M‐1, which grew in a monolayered sheet in F‐10 synthetic medium supplemented with 10% fetal bovine serum. T3M‐1 cells grew in rolling bottles at the velocity of 0.5 r.p.m. with about 22 hr. of population doubling time. When the culture reached confluency, it was incubated in a serum‐free medium supplemented with 1% bovine serum albumin. The conditioned medium was harvested every week, concentrated by Amicon PM‐10 membrane, and loaded on a Sephadex G‐75 column. The molecular weight of G‐CSF was estimated at about 30,000. This G‐CSF was stable over a pH range of 1.0 to 11.0 at 4°C for 21 hr. The CSF activity was destroyed by either trypsin or chymotrypsin, but resisted to RNase and DNase. A slight decrease in the activity was produced by treatment with neuramidase. G‐CSF stimulated granulocytic colony formation of human and mouse marrow cells. By using the roller bottle culture system, we could obtain more than 100 liters of cultured medium in a month, which was able to form about 150,000,000 colonies of human bone marrow cells. The recovery of the human G‐CSF activity from gel‐filtration column was very high (91.7%), and a large increase of specific activity was obtainable (13.3‐fold). This culture system is therefore expected to aid in the large‐scale preparation of human G‐CSF, thereby facilitating further studies on this granulopoietic factor.
Defective basal lamina formation by transformed mammary epithelial cells: A reduced effect of collagen on basal lamina (heparan sulfate‐rich) proteoglycan degradation
Authors: David Guido , Bernfield Merton
Published: February 2005

Invasive, spontaneously transformed mammary epithelial cells derived from the normal NMuMG cell line have lost the ability of their parental cells to respond in vitro to the presence of a collagen substratum by forming a continuous glycosaminoglycan (GAG)‐rich basal lamina. On collagen, the cells synthesize 35S‐GAG at the same rates, but the transformed cells accumulate less 35S‐GAG than the normal cells because a larger fraction of their newly synthesized 35S‐GAG is rapidly degraded. Chromatography of the 35SO4‐containing materials from cultures on collagen indicates that the reduced accumulation of 35S‐GAG by the transformed cells reflects less of a heparan sulfate‐rich proteoglycan fraction which has been found in the basal lamina. On plastic substrata, however, the normal and transformed cells have near identical rates of 35S‐GAG synthesis and degradation and they accumulate similar low amounts of the basal lamina proteoglycan fraction, which is rapidly degraded.

Thus, transformation appears to impair the ability of the cells to reduce basal lamina proteoglycan degradation in response to collagen. This impairment may prevent the neoplastic cells from forming a complete basal lamina and, therefore, allow local invasion.
Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum‐free medium
Authors: Giguère L. , Cheng J. , Gospodarowicz D.
Published: February 2005

Experimental conditions have been defined that allow bovine corneal endothelial (BCE) cells to grow in the complete absence of serum. Low density BCE cell cultures maintained on extracellular matrix (ECM)‐coated dishes and plated in the total absence of serum proliferate actively when exposed to a synthetic medium supplemented with high density lipoprotein (HDL 500 μg protein/ml), transferrin (10 μg/ml), insulin (5 μg/ml), and fibroblast (FGP) or epidermal growth factor (EGF) added at concentrations of 100 or 50 ng/ml, respectively. Omission of any of these components results in a lower growth rate and/or final cell density of the cultures. BCE cell cultures plated on plastic dishes and exposed to the same synthetic medium grow very poorly. The longevity of BCE cultures maintained on plastic versus ECM and exposed to serum‐free versus serum‐containing medium has been studied. The use of ECM‐coated dishes extended the life span of BCE cultures maintained in serum‐supplemented medium to over 120 generations, as compared to less than 20 generations for cultures maintained on plastic. Likewise, BCE cells maintained on ECM and exposed to a synthetic medium supplemented with optimal concentrations of HDL, transferrin, insulin, and FGF underwent 85 generations, whereas control cultures maintained on plastic could not be passaged. The enhancing effect of ECM on BCE cell growth and culture longevity clearly illustrates the importance of the cell substrate in the control of proliferation of these cells.
Mitogenic activity elaborated by macrophage‐like cell lines acts as competence factor(s) for BALB/c 3T3 cells
Authors: Wharton Walker , Gillespie G. Yancey , Russell Stephen W. , Pledger W. J.
Published: February 2005

The culture medium from several murine macrophage‐like cell lines contained a mitogenic activity that functioned synergistically with platelet‐poor plasma to induce DNA synthesis in quiescent density‐inhibited BALB/c 3T3 fibroblasts. This mitogenic activity was generated by P388D1 (and other established lines of) macrophage‐like cells that were cultured either in medium alone or in medium supplemented with platelet‐poor plasma. The amount of mitogenic activity produced was directly related to the length of time the macrophage‐like cells were maintained in the medium. Serum‐free medium conditioned by macrophage‐cells did not stimulate DNA synthesis in density‐inhibited 3T3 cells in the absence of plasma; however, a transient (4‐hr) exposure to serum‐free macrophage‐conditioned medium allowed quiescent cells to respond to plasma‐derived progression factors. The addition of plasma to 3T3 cells that had been treated with the macrophage‐conditioned medium brought about DNA synthesis after a 12‐hr lag. The mitogenic activity that was in macrophage‐conditioned medium bound to DEAE‐Sephadex and eluted in a single peak using a linear NaCl gradient. This macrophage‐derived competence factor was not mitogenic for lymphocytes and was clearly separated by DEAE‐Sephadex chromatography from the major peak of the previously described mitogenic monokine, Interleukin‐I (lymphocyte activating factor).
Mouse embryos contain polypeptide growth factor(s) capable of inducing a reversible neoplastic phenotype in nontransformed cells in culture
Authors: Proper Jacqueline A. , Bjornson Chris L. , Moses Harold L.
Published: February 2005

A growth‐factor‐like substance capable of inducing nontransformed mouse AKR‐2B, rat NRK, and EGF‐receptorless mouse NR6 cells to form progressively growing colonies in soft agar was identified in acid/ethanol extracts of 17‐day mouse embryos. This “mouse embryo factor” (MEF) is similar to previously described transforming growth factors in that it is capable of stimulating DNA synthesis and conferring a reversible transformed morphology on nontransformed cells in vitro. Passage of crude embryo extracts over a Bio‐Gel P‐60 column gave a major peak of soft agar growth‐stimulating activity in the 15,000 molecular weight range with a minor peak at about 22,000. This biological activity was sensitive to treatment with either trypsin or dithiothreitol, but was unaffected by heat (56°C for 30 minutes or 100°C for 3 minutes), indicating that the activity is due to a heat‐stable polypeptide(s) with disulfide bonds. Separation of these polypeptide(s) by chromatography on carboxymethyl cellulose revealed two peaks of soft agar growth‐stimulating activity which did not cochromatograph with a peak of epidermal growth factor receptor‐competing activity. The similarities of this mouse embryo‐derived growth factor to previously identified transforming growth factors suggest that both fetal development and neoplastic transformation may be affected by similar mechanisms.
Changes in phenotypic expression in embryonic and adult cells treated with 5‐azacytidine
Authors: Taylor Shirley M. , Jones Peter A.
Published: February 2005

We have previously shown that 5‐azacytidine (5‐Aza‐CR) induced the formation of biochemically differentiated myotubes, adipocytes, and chondrocytes in the mouse embryo cell line, C3H/10T1/2CL8 (10T1/2), and that the induction of the muscle phenotype was cell cycle specific. Here we show that the adipocyte phenotype is also induced maximally in cells treated during early S phase. During this period, the minimum treatment time required for the subsequent formation of myotubes was 5 min and the number of myotubes formed was dependent on treatment time. The incorporation of 14C‐5‐Aza‐CR into DNA during the cell cycle, however, was not enhanced during early S phase, suggesting that incorporation of 5‐Aza‐CR into specific DNA sequences synthesized during early S phase may be required for the expression of the new phenotypes. Single cells, obtained by plating cell suspensions into 16 mm wells at limiting dilution, were treated with 5‐Aza‐CR during S phase. The resulting clones showed a high frequency of phenotypic conversion, indicating that 5‐Aza‐CR did not act via a selective mechanism, and several of the clones were capable of expressing more than one phenotype. The cells required more than 2 division cycles after treatment with the analog for the expression of the muscle phenotype and the capacity to differentiate was retained for long periods of time in the absence of cell division. The adult mouse line, CVP3SC6, differentiated into functional striated muscle cells following treatment with 5‐Aza‐CR. The analog also caused oncogenic transformation in the adult line at the same concentration that was effective at inducing myogenic expression.
Simian virus 40 a gene function: Further characterization and growth of tsA transformed Chinese hamster cells
Authors: Robinson Christine C. , Lehman John M.
Published: February 2005

Chinese hamster embryo cells transformed with the tsA 58 mutant of Simian virus 40 express the transformed phenotype at the permissive temperature (33°C or 37°C) and a “normal” phenotype at the nonpermissive temperature (40.5°C). Immunofluorescence and immunoprecipitation of T antigens demonstrated that the “T” antigen (100 K) has an increase rate of synthesis and degradation at 40.5°C. However, the cells continue to replicate at the nonpermissive temperature when assayed by flow cytometry and autoradiography. This DNA synthesis was cellular, not viral, and not owing to an increase in DNA repair. When the cell cycle distributions of G1, S, and G2 + M were assayed by the fraction labeled mitoses method, no differences were evident at the permissive and nonpermissive temperature; however, the doubling time was lengthened at 40.5°C, the tsA transformed cells are cycling and dying. However, if the transformed cells are seeded onto monolayers of normal Chinese hamster cells at 40.5°C, the cells are growth arrested when measured by growth assays, flow cytometry, autoradiography, and immunofluorescence for T antigen. Therefore, growth arrest can be obtained in tsA 58 transformed Chinese hamster cells when cocultured with normal Chinese hamster cells.
Synergistic effect of purine derivatives on the toxicity of pyrazofurin and 6‐azauridine towards cultured mammalian cells
Authors: Gupta Radhey S. , Moffat Malcolm R. K.
Published: February 2005

A novel synergistic effect of several purine derivatives such as adenine, adenosine, hypoxanthine, and guanine on the toxicity of nucleoside analogs pyrazofurin and 6‐azauridine towards cultured Chinese hamster ovary (CHO) cells has been observed. The presence of the above purine derivatives enhanced the toxicity of pyrazofurin and 6‐azauridine, in a dose dependent manner. The growth inhibitory effects of these nucleoside analogs either alone or in combination with the purine derivatives were reversed by uridine and cytidine, providing evidence that the synergistic effect of the purine derivatives was exerted at the level of pyrimidine nucleotide biosynthesis. Studies with mutant cells lacking various purine phosphorylating enzymes show that phosphorylation of purine derivatives through reactions utilizing phosphoribosylpyrophosate (PRPP) is essential for observing the synergistic response. It is suggested that the above purine derivatives (including adenosine, via conversion to hypoxanthine) exert their synergistic effects by depleting the cellular pool of PRPP by two separate mechanisms (direct utilization and feedback inhibition of its synthesis), which as a result becomes rate limiting in the synthesis of orotidine monophosphate (OMP). The reduced levels of OMP, which is a competing substrate with pyrazofurin‐ and 6‐azauridine‐5'‐monophosphates for binding to the target enzyme OMP decarboxylase, could then account for the inhibition of the enzyme at lower concentrations of these analogs.
The relation between protein accumulation and cell cycle traverse of human NHIK 3025 cells in unbalanced growth
Authors: Rønning Øsytein W. , Seglen Per O.
Published: February 2005

Human NHIK 3025 cells, synchronized by mitotic selection, were given 2 mM thymidine, which inhibited DNA synthesis without reducing the rate of protein accumulation. After removal of the thymidine the cells proceeded towards mitosis and cell division, with an S duration 2 hours shorter than, but a G2 and M duration nearly identical to that of the control cells. If cycloheximide (1.25 m̈M) was present together with thymidine, no net protein accumulation took place during the treatment, and the subsequent duration of S, G2, and M was similar to that of the untreated cells. The shortening of S seen after treatment with thymidine alone would therefore indicate that the rate of DNA synthesis depended on the amount of some preaccumulated protein.

The postreplicative period in thymidine‐treated cells was lengthened by cycloheximide treatment although the protein content had already been doubled. This suggests that proteins required for the traverse of this part of the cell cycle might have to be synthesized after completion of DNA replication.

Shortly after removal of thymidine, the rate of protein accumulation declined markedly, indicating the existence of some mechanism for negative control of cell mass. In addition, the daughters of thymidine‐treated cells had their cell cycle shortened by 2 hours. As a result, the cells had returned to balanced growth already in the first cell cycle following the induction of unbalanced growth.

In conclusion, our experiments suggest that NHIK 3025 cells might require a minimum time in order to traverse the cell cycle, which is independent of cell mass.
A new model system for studying the phosphatidylinositol cycle
Authors: Monaco Marie E. , Lippman Marc E.
Published: February 2005

An early manifestation of the response of WRK‐1 rat mammary tumor cells to vasopressin is an increase in incorporation of (32P)Pi into phospholipids. Incorporation into all classes of phospholipids is stimulated; however, incorporation into phosphatidylinositol (PI) is increased to the greatest degree (3‐ to 10‐fold as compared with 1.3‐ to 2‐fold for the other phosholipids). Furthermore, increased incorporation into PI is accompanied by an increased rate of PI turnover; turnover rates of the other phospholipids are unaffected by vasopressin.
Characterization of neutral amino acid uptake by cultured epithelial cells from pig kidney
Authors: Sepúlveda Francisco V. , Pearson Jeremy D.
Published: February 2005

Two transport systems for neutral amino acids have been characterised in LLC‐PK1 cells. The first, which transport alanine in a sodium‐dependent manner, also mediates alanine exchange and is preferentially inhibited by serine, cysteine, and α‐amino‐n‐butyric acid. This system resembles the ASC system in Ehrlich ascites and some other cell types. There is only a small contribution of other systems to alanine uptake. The second, which transports leucine with no requirement for sodium and mediates leucine exchange, is blocked by 2‐aminonorbornane‐2‐carboxylic acid and hydrophobic amino acids. This system is similar to the L system described in other cell types. LLC‐PK1 cells retain several other features implying renal proximal tubule origin; our results thus suggest that these transport systems may be involved in the reabsorption of neutral amino acids by the nephron in vivo.
The role of vitamin E in cellular energy metabolism in cultured adrenocortical cells
Authors: Hornsby Peter J.
Published: February 2005

AC1 cells, a bovine adrenocortical cell clone, are rapidly arrested and killed by 2 mM aminooxyacetate, an inhibitor of transmination reactions. Toxicity of aminooxyacetate to cultured bovine adrenocortical cells was previously linked to a high ratio of capacity for oxidation of glutamine relative to pyruvate and was reversible by adding excess glutamine. The toxic effects of aminooxyacetate were shown in the present study to be completely prevented by the simultaneous addition of low concentrations (<100 nM) of d‐α‐tocopherol (vitamin E) or selenious acid or higher concentration of other tocopherols, antioxidants, and ubiquinones (coenzymes Q6 and Q10). When antioxidant analogs were tested, it was found that structural features of the molecules that increase antioxidant potency increased potency for prevention of aminooxyacetate toxicity. α‐Tocopherol‐supplemented AC1 cells were found to be significantly less sensitive than nonsupplemented cells to growth inhibition by rotenone but not by fluorocitrate or dinitrophenol. Dinitrophenol (100 m̈M) stimulated substrate oxidation fivefold but had relatively slight effects on growth, either with or without α‐tocopherol, indicating that limitation of the maximal activity of the tricarboxylic acid cycle and respiratory chain is probably not responsible for the sensitivity to aminooxyacetate and rotenone in cells not supplemented with α‐tocopherol. Sensitivity to growth inhibition by rotenone and the prevention by ubiquinones of aminooxyacetate toxicity suggest a restriction of electron flow at the NADH‐ubiquinone step. The resultant higher NADH/NAD+ ratio would result in a lowered capacity for metabolism of pyruvate with consequent dependence for tricarboxylic acid cycle function and energy production on 2‐oxoglutarate from glutamine by the transmination pathway. Vitamin E and other antioxidants may restore efficient function of ubiquinone by preventing side reactions involving lipid peroxidation. Selenium may have a similar effect as cofactor for glutathione peroxidase. A high ratio of capacity for oxidation of glutamine relative to pyruvate and accompanying sensitivity to aminooxyacetate may reflect a deficiency of vitamin E and selenium in adrenocortical cells in culture.
Effects of cyclophosphamide on murine bone marrow and splenic megakaryocyte‐CFC, granulocyte‐macrophage‐CFC, and peripheral blood cell levels
Authors: Yeager Andrew M. , Levin Francine C. , Levin Jack
Published: February 2005

The effect of cyclophosphamide (CY) on megakaryocytopoiesis in mice was examined with assays of megakaryocyte colony‐forming cells (Meg‐CFC) in bone marrow and spleen and simultaneous determinations of peripheral blood counts, after a single intraperitoneal dose (200 mg/kg) of CY. Significant rebound thrombocytosis (170% of normal) occurred at day 11 after injection with CY, although only modest preceding thrombocytopenia (70% of normal) was observed. After an initial 3–5‐day period of suppression, total megakaryocyte colony‐forming cells (Meg‐CFC) in both bone marrow and spleen of CY‐treated mice demonstrated rebound increases at 5 and 7 days, respectively, after administration of the drug. Granulocyte‐macrophage colony‐forming cells (GM‐CFC) exhibited alterations which were similar to those of Meg‐CFC, suggesting similar sensitivities of Meg‐CFC and GM‐CFC to CY. The increase in Meg‐CFC in both bone marrow and spleen preceded development of thrombocytosis by 4–6 days. This suggests that increased platelet counts in CY‐treated mice are attributable, at least in part, to alterations in feedback mechanisms which control megakaryocytopoiesis, with resultant stimulation of the megakaryocyte progenitor compartment.
Transport of cystine and cysteine and cell growth in cultured human diploid fibroblasts: Effect of glutamate and homocysteate
Authors: Bannai Shiro , Ishii Tetsuro
Published: February 2005

Human diploid fibroblasts take up cystine in the culture medium and the cystine is immediately reduced to cysteine in the cells. It is found that cysteine thus formed is rapidly released from the cells into the medium and accumulates there. The system transporting cysteine is convincingly similar to the ASC system described by Christensen et al. (1967). Since cysteine in the medium is sensitive to autoxidation and readily changes back to cystine, the uptake of cystine seems crucial to the cells. Inhibitors of cystine uptake, such as glutamate and homocysteate, potently reduce the intracellular and extracellular levels of cysteine. These inhibitors modify the cell growth depending upon the cystine concentration in the medium. They inhibit the growth when cystine concentration is physiological. An excessive concentration of cystine is in itself growth inhibitory, but this inhibitory action is antagonized by glutamate or homocysteate.
Dual pathways for epidermal growth factor processing after receptor‐mediated endocytosis
Authors: Miskimins W. Keith , Shimizu Nobuyoshi
Published: February 2005

The binding, internalization, intracellular translocation, and degradation of epidermal growth factor (EGF) were studied in mouse Swiss/3T3 fibroblasts under two different physiological conditions at 37°C. In serum‐containing medium the maximal level of cell‐bound EGF was maintained for at least 8 h without appreciable degradation in contrast to serum‐free conditions. These phenomena were correlated with a difference in the intracellular site to which the receptor‐bound EGF was delivered as studied using Percoll density gradients. In serum‐containing medium the majority of cell‐bound EGF was initially taken up into a Golgi‐like vesicle of density 1.046, corresponding to the marker galactosyl transferase, and then delivered to a population of vesicles with similar density as lysosomes (ϱ =1.068–1.110). A portion of the EGF became degraded and was released from the cell into the medium while the remainder stayed with the cells, intact, for a long period of time. In serum‐free medium, EGF became associated with a heterogeneous population of vesicles with a mean density of 1.050 which do not correspond to any of the marker enzymes for subcellular organelles for which we have tested (Golgi, endoplasmic reticulum, plasma membrane, lysosomes). It is then transferred to lysosome‐like vesicles (ϱ = 1.068–1.110). We therefore propose that EGF is processed through two separate endocytotic routes which are regulated by the cell depending upon its physiological state.
Effects of GM‐CSF deprivation on precursors of granulocytes and macrophages
Authors: Metcalf D. , Merchav S.
Published: February 2005

Culture of C57BL bone marrow cells in the absence of GM‐CSF led to a loss of recoverable granulocyte‐macrophage colony‐forming cells of 2% per hour. The rate of loss of progenitor cells in cultures of CBA fetal liver cells was 5–6% per hour. Surviving colony‐forming cells exhibited a normal responsiveness to GM‐CSF but generated smaller colonies than normal when subsequently stimulated by GM‐CSF. Transfer of washed individual day‐3 granulocyte‐macrophage colony cells to cultures lacking GM‐CSF indicated that most cells were unable to survive or proliferate in the absence of GM‐CSF. Death of transferred cells was rapid and invariable when the cells were from macrophage‐forming colonies. However some cells from 40–70% of granulocyte‐forming colonies were able to undergo one or two divisions in the absence of GM‐CSF. This phenomenon was seen most often with cells from colonies where matching colony cells exhibited a higher‐than‐average proliferative capacity in parallel stimulated cultures. The results indicate the difficulty that will be encoutered in obtaining valid metabolic dta from unstimulated populations of granulocyte‐macrophage precursor cells. The ability of some granulocyte precursor cells to exhibit limited proliferation following GM‐CSF deprivation suggests that significant amounts of GM‐CSF may be bound to or be internalized in some precursor cells and result in cell division in the absence of GM‐CSF from culture medium.
Down regulation and recovery of the epidermal growth factor receptor in serum supplemented versus defined medium
Authors: Kegel Mark , Tupper Joseph T.
Published: February 2005

The down regulation of surface membrane receptors for(125I) epidermal growth factor (EGF) has been evaluated in normal and SV40‐transformed human fibroblasts (WI38) under conditions of serum‐supplemented versus defined growth media. Both normal and transformed WI38 cells down regulate and recover the EGF receptor and these processes do not differ significantly in serum‐supplemented versus defined media. These data are in contrast to a recent study that reported that the HeLa cell does not down regulate the EGF receptor in defined media, whereas it does in serum‐supplemented media.
Isolation by a replica‐plating technique of chinese hamster temperature‐sensitive cell cycle mutants
Authors: Hirschberg Joseph , Marcus Menashe
Published: February 2005

Isolation of a wide variety of temperature‐sensitive (ts) cell cycle mutants in mammalian cells has previously proved to be a very difficult task. The various procedures used for the isolation of such mutants included a mutant enrichment step based on exposure of the cells to the restrictive temperatures in order to kill the growing wild‐type cells with agents that kill DNA‐synthesizing cells. Hence, these methods favored the isolation of ts mutants that do not lose viability rapidly at the restrictive temperatures, We have treated cells of the Chinese hamster established cell line E36 with the mutagen ethyl‐methane‐sulfonate (EMS) and used a replicaplating technique that we developed to screen the ts mutants for growth. This technique enabled us to recover all ts mutants for growth including the ts cell cycle mutants. Screening of the ts cell cycle mutants among the ts mutants for growth was performed by the flow microfluorimetry technique and the premature chromosome condensation technique. Our results show that 1.3% of the survivors of the mutagenic treatment are ts mutants for growth. Six of 84 ts mutants analyzed were found to be ts cell cycle mutants. They include ts mutants arrested in phases G1, S, and G2. Many of the ts mutants for growth including the ts cell cycle mutants arrested in S and G2 lose viability very fast when incubated at the restrictive temperature. As a consequence they could not have been isolated by any method that includes a mutant enrichment step based on the exposure of the cells to the restrictive temperature.
Decreased heme content and cessation of cell growth in cultured chick embryo fibroblasts in the presence of horse serum: Stimulation of heme synthesis and cell growth by iron
Authors: Verger Claude , Imbenotte José
Published: February 2005

A new spectrofluorometric method for heme quantitation in cultured fibroblasts is described. The method includes: (1) heme extraction by methanol/sulfuric acid, (2) partial purification of heme by a microchromatographic method, and (3) treatment of the purified heme by oxalic acid followed by fluorometric quantitation. Using this method, heme concentration was determined in chick embryo fibroblasts cultured in a medium supplemented with either 7% fetal bovine serum (FBS) or 10% horse serum (HS). In the presence of FBS, cultured cells actively divided and cells contained 34–55 pmol heme/mg protein. In contrast, cultures maintained in HS proliferated at a slower rate and contained 23–25 pmol heme/mg protein. The addition of 40 μM FeSO4 to cultures maintained in the presence of HS stimulated cell proliferation, and the cellular heme concentration increased to 37–51 pmol/ mg protein. These findings suggest that the cessation of growth in the presence of HS may be due to decreased heme content in the cells and that the stimulation of cell growth by iron is mediated by its stimulation of heme synthesis.