bluuebunny/crossref_metadata_2025 huggingface dataset:
Down‐modulation of epidermal growth factor receptor accompanies TNF‐induced differentiation of the DiFi human adenocarcinoma cell line toward a goblet‐like phenotype
Authors: Novotny‐Smith C. L. , Zorbas M. A. , McIsaac A. M. , Irimura T. , Boman Bruce M. , Yeoman L. C. , Gallick G. E.
Published: February 2005

Although the biologic response modifier tumor necrosis factor‐alpha (TNF) is a known differentiation Inducer in hematopoietic cells, its role in differentiation of other tissue types has yet to be elucidated. In the studies presented here, TNF treatment of the human rectal adenocarcinoma cell line, DiFi, elicits characteristics of early stage differentiating, mucin‐producing colonocytes. Not only are TNF‐treated DiFi cells growth‐inhibited by TNF, but they also display a unique morphology. Additionally, TNF treatment of DiFi cells enhances > fivefold the expression of high molecular weight mucin glycoproteins, as measured by [125I]‐wheat germ agglutinin (WGA) binding and the human milk fat globule‐1 (HMFG‐1) anti‐MUC1 antibody reactivity. The induction of these differentiation characteristics correlates with novel alterations in epidermal growth factor receptor (EGF‐R). Following 5‐day TNF treatment of DiFi cultures, EGF receptor levels, kinase autophosphorylation activity, and receptor tyrosine phosphorylation are reduced by > fourfold. The establishment of a model system in which goblet‐like cell characteristics and alterations in a growth factor receptor can be induced in vitro may be potentially useful in studying the underlying mechanisms of colonic epithelial cell proliferation and differentiation. © 1993 Wiley‐Liss, Inc.
Different patterns of apoptosis of HL‐60 cells induced by cycloheximide and camptothecin
Authors: Li Jianping Gong Xun , Darzynkiewicz Zbigniew
Published: February 2005

Cells of the human promyelocytic HL‐60 line, when treated with a variety of antitumor agents in the presence of the protein synthesis inhibitor cycloheximide (CHX), or with CHX alone, rapidly undergo apoptosis (“active cell death”). It is presumed, therefore, that such cells are “primed” to apoptosis in that no new protein synthesis is required for induction of their death. We have studied apoptosis of HL‐60 cells triggered by the DNA topoisomerase I inhibitor camptothecin (CAM) in the absence and presence of CHX and apoptosis induced by CHX alone. Two different flcw cytometric methods were used, each allowing us to relate the apoptosis‐associated DNA degradation to the cell cycle position. Apoptosis induced by CAM was limited to S phase cells, e.g., at a CAM concentration of 0.15 μM, nearly 90% of the S phase cells underwent apoptosis after 4 h. In contrast, apoptosis triggered by CHX was indiscriminate, affecting all phases of the cycle: ∼40% of the cells from each phase the cycle underwent apoptosis at 5 μM CHX concentration. When CAM and CHX were added together, the pattern of apoptosis resembled that of cycloheximide alone, namely, cells in all phases of the cycle in similar proportion were affected. Thus, CHX, while itself inducing apoptosis of a fraction of cells, protected the S phase cells against apoptosis triggered by CAM. Because CHX (5 μM) did not significantly affect the rate of cell progression through S phase, the observed protective effect was most likely directly related to inhibition of protein synthesis, rather than to its possible indirect effect on DNA replication. Furthermore, whereas apoptosis (DNA degradation) triggered by CAM was prevented by the serine protease inhibitor N‐tosyl‐L‐lysylchloromethyl ketone (TLCK), this process was actually potentiated by this inhibitor when induced by CHX. The present data indicate differences in mechanism of apoptosis triggered by CAM (and perhaps other antitumor drugs) as compared with CHX. Apoptosis caused by CHX may be unique in that it may not involve new protein synthesis. These data are compatible with the assumption that the loss of a hypothetical, rapidly turning over suppressor of apoptosis may be the trigger of apoptosis of HL‐60 cells treated with CHX, whereas de novo protein synthesis is required when apoptosis is triggered by other agents. © 1993 Wiley‐Liss, Inc.
Cell cycle activity and expression of prohibitin mRNA
Authors: Roskams A. Jane I. , Friedman Varda , Wood Carla M. , Walker Lisa , Owens Garrison A. , Stewart David A. , Altus Michael S. , Danner David B. , Liu Xiao‐Tie , McClung J. Keith
Published: February 2005

Prohibitin, a novel intracellular antiproliferative protein, blocks entry into the S phase of the cell division cycle when its mRNA is microinjected into normal fibroblasts or HeLa cells. To learn more about the interaction between prohibitin and the cell cycle, we studied the effect of microinjecting prohibitin mRNA at different points during the transition from G0 to S phase and analyzed prohibitin mRNA and protein levels in different parts of the cell cycle. The antiproliferative activity of microinjected prohibitin mRNA is high in G0/G1 and falls as cells approach S phase. Prohibitin mRNA and protein levels are high in G1, fall with S phase, rise again in G2, and fall in M. Together, these findings suggest that endogenous prohibitin contributes to the control of the G1 to S transition in cycling cells in a complex manner, which involves both a transcriptional and posttranslational mechanism. © 1993 Wiley‐Liss, Inc.
Transforming growth factor‐beta, basic fibroblast growth factor, and platelet‐derived growth factor‐BB interact to affect proliferation of clonally derived porcine satellite cells
Authors: Cook Douglas R. , Doumit Matthew E. , Merkel Robert A.
Published: February 2005

Transforming growth factor beta‐1 (1GF‐β) stimulated porcine satellite cell proliferation in basal serum‐free medium by 25%, but inhibited growth in serumcontaining medium by 58%. The effect of TGF‐β on cell proliferation in serumfree medium was examined in combination with the following human recombinant growth factors: platelet‐derived growth factor‐BB (PDGF), basic fibroblast growth factor (FGF), insulin‐like growth factor I (IGF‐I), and epidermal growth factor (EGF). TGF‐β inhibited PDGF‐stimulated proliferation, enhanced FGF‐stimulated proliferation, and had no effect on proliferation stimulated by IGF‐I. The response of satellite cells to EGF and TGF‐β in serum‐free medium was not different than TGF‐β alone. TGF‐β depressed proliferation stimulated by the following combinations of two growth factors: PDGF and IGF‐I, PDGF and EGF, PDGF and FGF, and IGF‐I and EGF. In combination with IGF‐I and FGF, TGF‐β did not affect proliferation. TGF‐β inhibited proliferation stimulated by the combination of PDGF, EGF, and IGF‐I, but had no effect on proliferation stimulated by combinations of three growth factors that included FGF. FGF stimulated proliferation in Minimum Essential Medium containing 10% porcine serum (MEM‐10% PS) by 13% above control. When the combination of TGF‐β and FGF was added to MEM‐10% PS, a 78% increase in proliferation was observed. Polyclonal antihuman PDGF‐AB (this form neutralizes PDGF‐AA, AB, and BB) reduced proliferation in MEM‐10% PS by 44%. The combination of TGF‐β and anti‐PDGF‐AB reduced proliferation by 59%, indicating the effects were not additive. These data indicate that: (1) FGF and TGF‐β interact to increase proliferation of clonally derived porcine satellite cells, and (2) the inhibitory effect of TGF‐β on proliferation of clonally derived porcine satelite cells can be primarily attributed to a reduction in the mitogenic effects of PDGF. © 1993 Wiley‐Liss, Inc.
Fibroblast growth factor, epidermal growth factor, insulin‐like growth factors, and platelet‐derived growth factor‐BB stimulate proliferation of clonally derived porcine myogenic satellite cells
Authors: Doumit Matthew E. , Cook Douglas R. , Merkel Robert A.
Published: February 2005

Clonally derived cultures of porcine skeletal muscle satellite cells were developed. The mitogenic effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin‐like growth factors (IGF‐I and ‐II), and platelet‐derived growth factors (PDGF‐AA and ‐BB) were examined. Individually, bFGF, IGFs, and PDGF‐BB stimulated proliferation of porcine satellite cells grown in basal serum‐free medium or Minimum Essential Medium containing 2% fetal bovine serum (MEM‐2% FBS). EGF stimulated proliferation in MEM‐2% FBS, but neither EGF nor PDGF‐AA, were mitogenic when added to serum‐free medium. The interactions among bFGF, [GF, IGF‐I], and PDGF‐BB were examined in serum‐free medium, using growth factor concentrations shown in dose‐response experiments to induce maximal proliferative responses (10 ng/ml bFGF, EGF and PDGF‐BB, and 50 ng/ml IGF‐I). The combination of bFGF and IGF‐I dramatically increased proliferation, and IGF‐I also synergized with EGF to increase proliferation. EGF, IGF‐I and bFGF interacted with PDGF‐BB to stimulate proliferation. With the exception of EGF and bFGF, combinations of two growth factors typically resulted in greater than additive responses. Simultaneous exposure of satellite cells to bFGF, PDGF‐BB, EGF, and IGF‐I produced a fivefold increase in DNA compared to cells grown in basal serum‐free medium. Elimination of EGF did not reduce the mitogenic response, yet removal of IGF‐I, bFGF, or PDGF‐BB reduced proliferation by ∼40, 20, and 10%, respectively. These mitogens are likely physiological regulators of porcine satellite cell activity. © 1993 Wiley‐Liss, Inc.
C‐FMS dependent HL‐60 cell differentiation and regulation of RB gene expression
Authors: Yen Andrew , Forbes Mary E. , Varvayanis Susi , Tykocinski Mark L. , Groger Richard K. , Platko Joseph D.
Published: February 2005

The dependence of induced myelomonocytic cell differentiation, and regulation of the RB tumor suppressor gene during this process, on the c‐fms gene product, the CSF‐1 lymphokine receptor, was determined in HL‐60 promyelocytic leukemia cells. Adding a monoclonal antibody with specificity for the c‐fms gene product to cells treated with various inducers of myelomonocytic or macrophage differentiation, including retinoic acid and 1,25‐dihydroxy vitamin D3, inhibited the rate of differentiation. During the period of inducer treatment usually preceding onset of differentiation, longer periods of antibody exposure caused greater inhibition of differentiation. In a stable HL‐60 transfectant overexpressing the CSF‐1 receptor at the cell surface due to a constitutively driven c‐fms trans gene, the rate of differentiation was enhanced compared to the wild type cell, consistent with a positive regulatory role for the CSF‐1 receptor. The anti‐fms antibody caused much less inhibition of differentiation in the transfectants than in wild type cells, consistent with a larger number of receptors causing reduced sensitivity. During the induced metabolic cascade leading to differentiation, the typical early down regulation of RB gene expression was inhibited by the antibody. The antibody itself caused an increase in RB expression per cell, which offset the decrease normally caused by differentiation inducers (1,25‐dihydroxy vitamin D3 and retinoic acid). The changes in RB expression preceded changes in the RB protein to the hypophosphorylated state. Most of the RB protein in proliferating cells was phosphorylated and no significant accumulation of hypophosphorylated RB protein occurred until after onset of GO arrest. Thus the metabolic cascade leading to myelomonocytic differentiation of HL‐60 cells appears to be driver by a function of the c‐fms protein. Inhibiting that process by attacking this receptor impedes differentiation and also compromises the early down regulation of RB tumor suppressor gene expression which normally precedes differentiation. These findings provide additional support for a potential role for down regulating RB expression in promoting cell differentiation, and suggest the possibility that RB may be either a target or intermediate mediator of CSF‐1 actions. © 1993 Wiley‐Liss, Inc.
Relative roles of Na+/H+ exchange and vacuolar‐type H+ ATPases in regulating cytoplasmic pH and function in murine peritoneal macrophages
Authors: Swallow Carol J. , Grinstein Sergio , Sudsbury Rae A. , Rotstein Ori D.
Published: February 2005

Two distinct mechanisms have been shown to mediate cytoplasmic pH (pH i) recovery in acid‐loaded peritoneal macrophages (Mψs): Na+/H+ exchange and H+ extrusion by vacuolar‐type (V‐type) H+ ATPases. The present studies examined the relative roles of these two systems in maintaining pHi and cell function. Measurements of Mψ pHi and superoxide (O2−) production in response to stimulation with 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) were made at physiological or acidic extracellular pH (pHo) levels. The V‐type H+ ATPase inhibitor, bafilomycin A1, and potent Na+/H+ exchange inhibitor, N‐ethyl‐N‐propylamino amiloride (EPA), were used to examine the contributions of these ion transporters to pHi regulation and cell function. At pHo 7.35, the complementary activities of the Na+/H+ antiport and the V‐type H+ ATPase mediate pHi homeostasis. At pHo 6.7, maintenance of pHi depends primarily on H+ ATPase activity: bafilomycin A1 reduced pHi from 6.8±0.02 in control cells to 6.59±0.01 (P<0.01) while EPA was without effect. The functional importance of V‐type H+ ATPase‐activity in preserving pHi homeostasis at acidic extracellular oH levels was reflected by the impairment of O2− production at pHo 6.70 when H+ ATPase activity was inhibited: bafilomycin A1 reduced O2− production from 13.9±1.0 to 9.3±0.6 nmoles/106 cells/40 min, in control and bafilomycin A1‐treated cells, respectively (P≤0.05), while EPA had no effect. In subsequent studies, pHi was independently manipulated using the ionophore nigericin. Lowering pHi from 6.80 to 6.60 reduced O2− production from 15.3±1.8 to 9.8±1.6 nmoles/106 cells/40 min (P≤0.05), indicating that the cytoplasmic acidification resulting from inhibition of H+ ATPases at low pHo could account for the associated impairment of O2− production. In a more profoundly acidic environment (pHo 6.35), H+ ATPases remained active in regulating pHi, but could not preserve a sufficiently physiological pHi to supprt respiratory burst activity. V‐type H+ ATPases constitute the dominant mechanism by which the pHi of peritoneal Mψs is maintained in an acidic extracellular environment. © 1993 Wiley‐Liss, Inc.
Effects of mitogens and co‐mitogens on the formation of small‐cell colonies in primary cultures of rat hepatocytes
Authors: Mitaka Toshihiro , Norioka Ken‐Ichi , Nakamura Toshikazu , Mochizuki Yohichi
Published: February 2005

Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum‐free Dulbecco's modified Eagle's medium containing 10 mM nicotinamide and 10 ng/ml of epidermal growth factor (EGF), acidic and basic fibroblast growth factors (FGF), hepatocyte growth factor (HGF), or transforming growth factor‐α (TGF‐α). Every colony consisted of cells that each had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically the cells induced by any mitogen were stained with albumin, transferrin, cytokeratin‐8 and ‐18. But these cells expressed neither cytokeratin‐7 nor‐19. When 6 × 105 cells were plated on 35‐mm dishes, about 15 colonies per 1,000 attached cells were observed in the cultures treated with EGF, HGF, and TGF‐α. Although FGFs could also induce colonies, their number was less than half of the number induced by EGF. Furthermore, the numbers of colonies induced by the combinations of EGF + HGF, EGF + TGF‐α, and HGF + TGF‐α were not different from those of the colonies induced by each mitogen alone. To examine the ability of co‐mitogenic factors to induce small‐cell colonies, angiotensin‐II, insulin‐like growth factor‐I, norepinephrine, tumor necrosis factor, and vasopressin were used. In the cells cultured without EGF, these co‐mitogens neither stimulated DNA synthesis nor induced colonies. On the other hand, in cells cultured with both EGF and each co‐mitogen, although the DNA synthesis of the hepatocytes was enhanced, the number of colonies detected was not significantly different from the number which EGF alone could induce. These results showed that the small‐cell colonies in primary cultures of rat hepatocytes were inducible by EGF, HGF, TGF‐α, or FGFs and that the co‐mitogens did not influence the formation of the small‐cell colonies. © 1993 Wiley‐Liss, Inc.
Lipid synthesis and secretion by primary cultures of rat mammary epithelial cells
Authors: Rohlfs Elizabeth M. , Louie Dexter S. , Zeisel Steven H.
Published: February 2005

Lipid synthesis and secretion was measured in primary rat mammary epithelial cells cultured on basement matrix in medium supplemented with lactogenic hormones. The cells grew and differentiated to form alveolar‐like structures reminiscent of lactating mammary gland. They synthesized abundant triacylglycerol, containing fatty acids characteristic of rat milk (C10:O‐C14:0), using 14C‐glucose, 14C‐oleic acid or 14C‐glycerol as precursors. Basal levels of triacylglycerol secretion were measured using 14C‐oleic acid labeling; 1.3±0.3% of the labeled cellular triacylglycerol was secreted into the medium in 24 hours. Secreted lipid droplets were surrounded by a bilayer membrane with an electron‐dense inner coat characteristic of fat globules secreted by the mammary gland. The rate of triglycerol secretion was increased to 998±98% of control (P<0.01) by the addition of phorbol 12‐myristate 13‐acetate (PMA) in combination with staurosporine, a protein kinase inhibitcn. Several other protein kinase inhibitors, when combined with PMA, also markedly stimulated secretion. Effective protein kinase inhibitors included sphingosine (has diverse cellular effects including the inhibition of protein kinase C; 13‐fold increase in secretion), and KT5823 (a cGMP dependent protein kinase inhibitor; 5‐fold increase). KT5720 (a cAMP‐dependent protein kinase inhibitor) did not alter secretion. Kinase inhibitors were effective only in the presence of a phorbol ester. 4α‐phorbol‐12,13‐didecanoate, a phorbol ester which does not activate protein kinase C (PKC), could substitute for PMA. Lipid release was not mediated by disruption of cell‐cell tight junctions, as EGTA did not release lipid. Based on these observations we suggest that two signals are needed to enable or stimulate lipid secretion in cultured rat mammary epithelial cells: (1) inhibition of a protein kinase and (2) a PKC‐independent effect of phorbol ester. We have, for the first time, characterized a cell culture model suitable for studying lipid synthesis and secretion by mammary epithelial cells. © 1993 Wiley‐Liss, Inc.
Superoxide‐mediated lysis of erythrocytes: The role of colloid‐osmotic forces
Authors: Zhou Ming‐Jie , Petty Howard R.
Published: February 2005

Although superoxide anions are a well‐known mediator of cytotoxicity, their mechanism of target cell lysis is not clearly understood. In the present study we have used an exogenous source of superoxide to study erythrocyte cytolysis. RBC lysis was studied in buffers containing the cations Li+, Na+, K+, Rb+, and Cs+; superoxide anions were produced and available in these buffers. During this model superoxide‐dependent cytolytic process, erythrocytes underwent a shape change from biconcave disk to sphere as shown by scanning electron microscopy. Soret band transmitted light microscopy has confirmed this shape change and shown that it precedes cytosolic oxidation. This evidence is consistent with a colloid‐osmotic type lytic mechanism. Erythrocyte lysis was studied by 51Crrelease and light scattering methods. Superoxide‐mediated target cytolysis was characterized by: (1) a sigmoidal dose‐response curve and (2) a lag time in cytolysis after superoxide addition in kinetic light scattering experiments. The efficacy of cytolysis followed the rank order Cs+ > Rb+ > Na+, Li+ > sucrose = raffinose, which provides additional support for a colloid‐osmotic lytic mechanism. Furthermore, the rank order potency correlates with the cations' hydration numbers. We suggest that oxidative events trigger the formation of colloid‐osmotic pores ∼I nm in diameter. © 1993 Wiley‐Liss, Inc.
Flow cytometry reveals different lag times in rapid cytoplasmic calcium elevations in human neutrophils in response to N‐formyl peptide
Authors: Elsner Jörn , Norgauer Johannes , Dobos Gustav J. , Emmendörffer Andreas , Schöpf Erwin , Kapp Alexander , Roesler Joachim
Published: February 2005

Flow cytometric analyses were performed to study intracellular single‐cell calcium transients ([Ca2+]i) in suspended human neutrophils during the initial phase of N‐formyl peptide stimulation. Thereby, two neutrophil populations became apparent. Early maximally Ca2+‐responding (high fluorescence) neutrophils and not‐yet Ca2+‐responding (low fluorescence) neutrophils, but no neutrophils with intermediate levels of [Ca2+]i, were detected. Within 7 s the number of low fluorescence neutrophils decreased and the number of high fluorescence neutrophils increased maximally. This suggests that [Ca2+]i transients occurred abruptly in individual neutrophils within a time interval below 1 s. At lower N‐formyl peptide concentrations the lag times of individual neutrophils and the interval time of maximal activation of the [Ca2+]i‐responding neutrophil population increased, however the percentage of [Ca2+]i‐responding cells decreased. Surprisingly, no influence of the N‐formyl peptide concentration on the [Ca2+]i‐induced fluorescence signal of the individual cell was observed: it was always in an almost maximal range or not responding. In parallel, binding studies performed with fluorescein‐labeled N‐formyl peptide revealed that the heterogeneity of [Ca2+]i‐responding cells cannot be explained by different receptor occupancy. In summary, this study demonstrates that [Ca2+]i transients induced by N‐formyl peptides in suspended individual human neutrophils occur very rapidly in an almost “all‐or‐none manner” and that the mean increasing fluorescence signal of a calcium indicator within a whole neutrophil population results from varying lag times of the individual cells, rather than from the mean simultaneous progress of many cells. © 1993 Wiley‐Liss, Inc.
Human T lymphocytes synthesize the 92 kDa type IV collagenase (gelatinase B)
Authors: Weeks Benjamin S. , Schnaper H. William , Handy Michael , Holloway Eva , Kleinman Hynda K.
Published: February 2005

In order for T cells to exit the circulatory system, traverse the endothelial basement membrane, and arrive in target tissues, these cells must attach to and degrade basement membrane proteins. 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) has been shown to stimulate lymphoid cell adhesion to basement membrane components. We have used TPA to study the ability of human lymphoid cells to secrete type IV collagenases, enzymes capable of degrading basement membrane proteins. Here, we found that human primary T cells and H‐9 lymphoid cells synthesize the 92 kDa type IV collagenase (gelatinase B) and TPA stimulates the synthesis and secretion of this protease. Peak TPA‐stimulated gelatinase B secretion and mRNA accumulation were observed 9 hours after TPA treatment, while the peak adhesion to type IV collagen was observed only 3 hours after TPA treatment. The protein kinase C inhibitor, H‐7, inhibited TPA‐stimulated gelatinase B secretion. Both the primary T cells and H‐9 lymphoid cells also expressed the mRNA for the tissue inhibitor of metalloproteinase‐1 (TIMP‐1). These data demonstrate that TPA ‐ stimulated lymphoid cells adhere to type IV collagen and subsequently synthesize and secrete gelatinase B and TIMP‐1. We conclude that lymphoid cell extravasation may involve cellular employment of adhesion mechanisms prior to degradation of the matrix, which is similar to the process of extravasation used by metastatic cells. © 1993 Wiley‐Liss, Inc.
Insulin‐like growth factor‐binding protein enhancement of insulin‐like growth factor‐i (IGF‐I)–mediated DNA synthesis and IGF‐I binding in a human breast carcinoma cell line
Authors: Chen Jian‐Chyi , Shao Zhi‐Ming , Sheikh M. Saeed , Hussain Arif , Leroith Derek , Roberts Charles T. , Fontana Joseph A.
Published: February 2005

The insulin‐like growth factors (IGFs) are potent mitogens for malignant cell proliferation. The majority of secreted IGFs are bound to specific IGF‐binding proteins (IGFBPs) that are secreted by a large number of cells. These proteins may either inhibit or enhance IGF actions. Breast carcinoma cells secrete a variety of IGFBPs. We have previously demonstrated that retinoic acid (RA) inhibition of IGF‐l– stimulated MCF‐7 cell proliferation is associated with increased IGFBP‐3 levels in the conditioned media. We therefore investigated the effect of recombinant IGFBP‐3 as well as IGFBP‐2, ‐4 and ‐5 on IGF‐l stimulation of DNA synthesis and IGF‐I binding in the MCF‐7 human breast carcinoma cell line. IGFBP‐2 and ‐3 enhanced IGF‐l stimulation of DNA synthesis in MCF‐7 cells while IGFBP‐4 and ‐5 had no effect. Transfection of MCF‐7 cells with an IGFBP‐3 expression vector resulted in the enhanced secretion of IGFBP‐3 with an accompanying increase in IGF‐l binding as well as increased cell proliferation upon treatment of the cells with IGF‐l. IGF‐l preincubation of MCF‐7 cells transfected with control pSVneo plasmids results in cells refractory to further IGF‐l stimulation of thymidine incorporation while IGF‐l continues to stimulate [3H]‐thymidine incorporation in IGFBP‐3–transfected MCF‐7 cells, suggesting that IGFBP‐3 protects the cells from IGF‐l–mediated down regulation of its receptor. Therefore, IGFBP‐3 secreted by MCF‐7 cells can enhance IGF‐l stimulation of DNA synthesis, increase IGF‐l binding to these cells, and prevent IGF‐l–induced desensitization of its own receptor, suggesting that IGFBP‐3 plays a significant role in IGF‐l–mediated breast carcinoma proliferation. © 1994 Wiley‐Liss, Inc.
Migratory interaction of amphibian epidermal cells with components of the basement membrane
Authors: Donaldson Donald J. , Mahan James T. , Tsilibary Effie C. , McCarthy James B. , Dixit Saryu N. , Chung Albert E.
Published: February 2005

In adult newts, basal epidermal cells adjacent to a fresh wound move toward the damaged area by migrating over the epidermal basement membrane. In an attempt to determine which basement membrane components mediate this migration, small pieces of glass coated with various natural matrices, purified proteins, or fragments of proteins were implanted into skin wounds such that epidermal cells attempting to form a wound epithelium would encounter the implants. Laminin derived from a cell line (M1536‐B3) that produces no type IV collagen was inactive as a migration substrate. Migration on recombinant entactin was somewhat better than on laminin but was still only ∼ 14% of that on type I collagen. M15 matrix, a laminin and entactin‐containing product of M1536‐B3 cells, was no better than entactin alone. Type IV collagen was an excellent substrate, producing slightly more migration than corresponding concentrations of type I collagen at nearly all concentrations tested. Migration on type IV lacking the NC1 domain was at least as good as on intact type IV. All the activity in type IV was present in a 95 kD fragment (al (IV)95) from the carboxy terminal two‐thirds of the α1 chain. Approximately 60% of the activity on β1(IV)95 was obtained on implants coated with a 110 amino acid fragment of the α1 chain derived from the carboxy terminal half of α1(IV)95. Adding the synthetic peptide, arg‐gly‐asp‐ser (RGDS) to the medium, biocked migration on fibronectin‐coated implants but had no effect on implants coated with type IV, suggesting that migration on type IV involves different cell surface receptors than those mediating migration over fibronectin. Matrigel, a commercial product containing most basement membrane components, was a poor migration substrate. Thus if type IV mediates basal cell migration toward a wound in vivo, there may have to be some alterations in basement membrane structure to allow epidermal receptors to access type IV active site(s). © 1994 Wiley‐Liss, Inc.
Increased ambient pressure stimulates proliferation and morphologic changes in cultured endothelial cells
Authors: Sumpio Bauer E. , Widmann Mark D. , Ricotta Joseph , Awolesi Mark A. , Watase Makoto
Published: February 2005

Very little is known about the effects of pressure within the vascular system on EC phenotype. To study this, bovine aortic EC were seeded on rat type I collagen plates (2,000/cm2) and allowed to attach for 24 hours. The cells were exposed to either atmospheric, 40, 80, or 120 mm Hg pressure by placing them in a plexiglass pressure chamber loaded with 5% CO2/air and maintained at 37°C inside an incubator. Chamber pressure was continuously monitored with an amplified voltage transducer connected to a digital monitor. EC were maintained in DMEM supplemented with 10% calf serum and substrates for up to 9 days. The results indicate that EC proliferation is influenced by their ambient pressure. EC subjected in vitro to pressures comparable to mean systemic blood pressures had a significant increase in cell number compared to EC exposed to atmospheric pressures. EC were elongated and appeared to align randomly. We hypothesize that the systemic pressure which the endothelium is exposed to in vivo may have a significant regulatory influence on the ability of the endothelium to proliferate which may affect the endothelial cell response to injury. © 1994 Wiley‐Liss, Inc.
Upregulation of IGF1, IGF1‐receptor, and late growth related genes in ventricular myocytes acutely after infarction in rats
Authors: Reiss Krzysztof , Meggs Leonard G. , Li Peng , Olivetti Giorgio , Capasso Joseph M. , Anversa Piero
Published: February 2005

To determine the effects of acute myocardial infarction on the expression of insulin‐like growth factor1 (IGF1) and insulin‐like growth factor1 receptors (IGF‐1R) on the surviving myocytes of the left and right ventricles, large infarcts were produced in rats and the animals sacrificed 2 days later. Hemodynamic measurements of left and right ventricular pressures, +dP/dt and ‐dP/dt, and central venous pressure documented that coronary occlusion was associated with a severe impairment of cardiac function. By employing reverse transcriptase polymerase chain reaction (RTPCR), a low level of expression of IGF‐1R mRNA was detected in myocytes from sham‐operated rats. Acute myocardial infarction was found to enhance by nearly twofold the message for IGF‐1R in viable myocytes biventricularly. Moreover, IGF1 mRNA increased 4.3‐fold and 9.4‐fold in left and right myocytes, respectively. In order to establish whether the upregulation of IGF1 and IGF‐1R with infarction was coupled with induction of late growth related genes, which are known to be implicated in DNA replication and mitotic division, proliferating cell nuclear antigen (PCNA) and histone‐H3 expression was assessed by Northern blot and RTPCR. The level of expression of PCNA mRNA was found to be increased 3.9‐fold and 2.4‐fold in left and right myocytes, respectively, from infarcted hearts. Corresponding increments in histone‐H3 mRNA were 25.5‐fold and 5.3‐fold, respectively. However, PCNA protein as detected by immunoperoxidase staining was restricted to a limited number of myocyte nuclei adjacent to the necrotic myocardium of the left ventricle. In conclusion, acute myocardial infarction is associated with enhanced expression of IGF1 and IGF‐1R on stressed myocytes, and this phenomenon may activate genes essential for DNA synthesis, possibly affecting myocyte growth. These processes may be fundamental for the reconstitution of tissue mass and amelioration of function after infarction. © 1994 Wiley‐Liss, Inc.
Alterations in the cyclic AMP signal transduction pathway regulating ribonucleotide reductase gene expression in malignant H‐ras transformed cell lines
Authors: Hurta Robert A. R. , Wright Jim A.
Published: February 2005

Ribonucleotide reductase is a highly regulated activity responsible for reducing ribonucleotides to deoxyribonucleotides, which are required for DNA synthesis and DNA repair. We have tested the hypothesis that malignant cell populations contain alterations in signal pathways important in controlling the expression of the two genes that code for ribonucleotide reductase, R1 and R2. A series of radiation and H‐ras transformed mouse 10T1/2 cell lines with increasing malignant potential were exposed to stimulators of cAMP synthesis (forskolin and cholera toxin), an inhibitor of cAMP degradation (3‐isobutyl‐1‐methylxanthine) and a biologically stable analogue of cAMP (8‐bromo‐cAMP). Dramatic elevations in the expression of the R1 and R2 genes at the message and protein levels were observed in malignant metastatic populations, which were not detected in the normal parental cell line or in cells capable of benign tumor formation. These changes in ribonucleotide reductase gene expression occurred without any detectable modifications in the rates of DNA synthesis, showing that they were regulated by a novel mechanism independent of the S phase of the cell cycle. Furthermore, studies with forskolin (a stimulator of the protein kinase A signal pathway) and the tumor promoter 12–0‐tetradecanoylphorbol‐13‐acetate (a stimulator of the protein kinase C signal pathway), alone or in combination, indicated that their effects on R1 and R2 gene expression in a highly malignant cell line were greater than when they were tested individually, suggesting that the two pathways modulating R1 and R2 gene expression can cooperate to regulate ribonucleotide reduction, and interestingly this can occur in a synergistic fashion. Also, a direct relationship between H‐ras expression and ribonucleotide reductase gene expression was observed; analysis of forskolin mediated elevations in R1 and R2 message levels closely correlated with the levels of H‐ras expression in the various cell lines. In total, these studies demonstrate that ribonucleotide reductase expression is controlled by a complex process, and malignant ras transformed cells contain alterations in the regulation of signal transduction pathways that lead to novel modifications in ribonucleotide reductase gene expression. This signal mechanism, which is aberrantly regulated in malignant cells, may be related to regulatory pathways involved in determining ribonucleotide reductase expression in a S phase independent manner during periods of DNA repair. © 1994 Wiley‐Liss, Inc.
Synthesis, degradation, and subcellular localization of proteins encoded by the primary response genes TIS7/PC4 and TIS21/PC3
Authors: Varnum Brian C. , Reddy Srinivasa T. , Koski Raymond A. , Herschman Harvey R.
Published: February 2005

Murine TIS7 and TIS21 cDNAs were cloned from phorbol ester‐induced Swiss 3T3 cells. The cognate rat cDNAs. PC4 and PC3, were cloned from nerve growth factor (NGF)‐treated PC12 pheochromocytoma cells. The TIS7/PC4 and TIS21/PC3 primary response genes are rapidly and transiently induced in response to serum, phorbol esters, and polypeptide growth factors in quiescent Swiss 3T3 cells and by NGF and other ligands in PC12 cells. In both 3T3 and PC12 cells the appearance of the TIS21/PC3 message precedes that of TIS7/PC4 message following ligand stimulation, suggesting that the TIS21/PC3 protein is likely to be synthesized more rapidly than the TIS7/PC4 protein. Using antisera prepared against recombinant TIS21 and TIS7 proteins, we find that the TIS21/PC3 protein is, indeed, synthesized more rapidly than the TIS7/PC4 protein following stimulation in both 3T3 and PC12 cells. In addition, “pulse‐chase” experiments demonstrate that the TIS21/PC3 protein is degraded much more rapidly than the TIS7/PC4 protein. The sequences of the predicted PC3 and PC4 proteins have lead to the speculation that these two proteins may both be secreted from cells following stimulation. The PC4 protein is reported to have some sequence similarity to interferons. The TIS21/PC3 protein contains a presumptive leader sequence. Using our antisera to the recombinant proteins, however, we cannot detect secretion of radiolabelled TIS7/PC4 or TIS21/PC3 protein. Immunohistochemical and subcellular fractionation experiments suggest that the TIS7 protein is a membrane associated, non‐nuclear intracellular protein. The TIS21 protein, in contrast, is' a non‐nuclear, soluble intracellular protein. © 1994 Wiley‐Liss, Inc.
Murine temperature‐sensitive DNA polymerase α mutant displays a diminished capacity to stimulate DNA synthesis in senescent human fibroblast nuclei in heterokaryons at the nonpermissive condition
Authors: Pendergrass William R. , Saulewicz Anthony C. , Hanaoka Fumio , Norwood Thomas H.
Published: February 2005

We have investigated the capacity of a murine cell line with a temperature‐sensitive (ts) mutation in the DNA polymerase α (Pola) locus and a series of ts non‐Pola mutant cell lines from separate complementation groups to stimulate DNA synthesis, in senescent fibroblast nuclei in heterokaryons. In the Pola mutant × senescent heterodikaryons, both human and murine nuclei display significantly diminished levels of DNA synthesis at the restrictive temperature (39.5°C) as determined by [3H]thymidine labeling in autoradiographs. In contrast, all of the non‐Pola mutants, as well as the parental (wild‐type) murine cells, induced similar levels of DNA synthesis in both parental nuclei at the nonpermissive and permissive temperatures. Similarly, young human fibroblasts are also able to initiate DNA synthesis in heterokaryons with the ts Pola mutant at the two temperatures. In order to determine if complementation of the non‐Pola mutants requires induction of serum responsive factors in the senescent cells, fusion studies of similar design were conducted with young and old human fibroblasts incubated in low serum (0.2%) for 48 hr prior to and after cell fusion. Again, a diminished level of DNA synthesis was observed at 39.5°C in the Pola mutant x senescent cell heterokaryons. In these low‐serum studies, both parental nuclei in the Pola x young cell heterokaryons and the human nuclei in heterokaryons with one of the non‐Pola mutants (FT107) also displayed diminished levels of DNA synthetic activity. All of the other mutants are able to support similar levels of synthetic activity at both temperatures in the presence of reduced serum. The nature of the mutation in three of the non‐Pola lines has not been determined but, like the Pola mutant cells, are inhibited in the G1 phase of the cell cycle when incubated at the nonpermissive temperature (39.5°C). The fourth non‐Pola mutant line is known to have at least one ts mutation in the cdc2 gene and is inhibited in the G2 phase when exposed to 39.5°C. These results suggest that there may be a functional deficiency of pol α in senescent human fibroblasts, and this replication factor may be one of the rate‐limiting factors involved in loss of the capacity to initiate DNA synthesis in senescent cells. © 1994 Wiley‐Liss, Inc.
Requirement for receptor‐bound urokinase in plasmin‐dependent cellular conversion of latent TGF‐β to TGF‐β
Authors: Odekon Lale E. , Blasi Francesco , Rifkin Daniel B.
Published: February 2005

The role of receptor‐bound urokinase‐type plasminogen activator (uPA) in cellular activation of latent transforming growth factor‐beta (LTGF‐β) was investigated in a model system of mouse LB6 cells transfected with either a human uPA receptor cDNA (LhuPAR+). a human prouPA cDNA (LhuPA), or a control neomycinresistance cDNA (Lneo). When LhuPAR+ cells were co‐cultured with LhuPA cells, the plasmin‐dependent fibrinolytic activity generated was more than that observed in either homotypic cultures with fivefold greater number of LhuPA cells or co‐cultures containing LhuPA and Lneo cells instead of the LhuPAR+ cells. The preferential activation of TGF‐β by co‐cultures with the greatest plasmin‐generation potential, LhuPAR+ and LhuPA cells, was confirmed by three independent bioassays. In the first assay, a 48% decrease in PA activity, a measure of active TGF‐β production, was observed with BAE cells treated with conditioned medium (CM) from co‐cultures of LhuPA and LhuPAR+ cells. Inclusion of neutralizing antibodies to TGF‐β abrogated the inhibitory effect of CM on PA activity demonstrating that the inhibitory molecule was TGF‐β. Addition of the amino terminal fragment of uPA (ATF) or omission of plasminogen from co‐cultures blocked both the fibrinolytic activity and the generation of TGF‐β activity in the CM. In the second assay, CM from co‐cultures of LhuPA and LhuPAR+ cells inhibited the migration of BAE cells in a wound assay. Controls with anti‐TGF‐β IgG indicated that the inhibition was due to TGF‐β. In the third assay, proliferation of mink lung epithelial cells was inhibited by CM generated by co‐cultures of LhuPA and LhuPAR+ cells as compared to CM from the same cells cultured in the absence of plasminogen or to CM from a co‐culture of LhuPA with LhuPAR− cells. Excess mannose‐6‐phosphate (M6P) blocked the generation of TGF‐β as assayed by both the BAE migration and PA assays, presumably because it interfered with cellsurface localization of LTGF‐β. Additionally, small numbers of LhuPA and LhuPAR+ cells co‐cultured with BAE cells inhibited the BAE cell PA activity via the paracrine action of TGF‐β. These results support the conclusion that plasmindependent activation LTGF‐β by LB6 cells is promoted by the surface localization of uPA by its receptor. © 1994 Wiley‐Liss, Inc.
Role of serum in the developmental expression of alkaline phosphatase in MC3T3‐E1 osteoblasts
Authors: Yohay Daniel A. , Zhang Jian , Thrailkill Kathryn M. , Arthur John M. , Quarles L. Darryl
Published: February 2005

MC3T3‐E1 cells in culture exhibit a temporal sequence of development similar to in vivo bone formation. To examine whether the developmental expression of the osteoblast phenotype depends on serum derived factors, we compared the timedependent expression of alkaline phosphatase (ALP)‐a marker of osteoblastic maturation‐ in MC3T3‐E1 cells grown in the presence of fetal bovine serum (FBS) or resin/charcoal‐stripped (AXC) serum. ALP was assessed by measuring enzyme activity, immunoblotting, and Northern analysis. Growth of MC3T3‐E1 cells in FBS resulted in the programmed upregulation of alkaline phosphatase (ALP) post‐proliferatively during osteoblast differentiation. In the presence of complete serum, actively proliferating cells during the initial culture period expressed low ALP levels consistent with their designation as pre‐osteoblasts, whereas postmitotic cultures upregulated ALP protein, message, and enzyme activity. In addition, undifferentiated early cultures of MC3T3‐E1 cells were refractory to forskolin (FSK) stimulation of ALP, but became forskolin responsive following prolonged culture in FBS containing media. In contrast, MC3T3‐E1 cells grown in AXC serum displayed limited growth and failed to show a time‐dependent increase in alkaline phosphatase. Neither the addition of IGF‐I to AXC serum to augment cell number or plating at high density restored the time‐dependent upregulation of alkaline phosphatase. Cells incubated in AXC serum for 14 days, however, though expressing low alkaline phosphatase levels, maintained the capacity to upregulate ALP after FBS re‐addition or forskolin activation of cAMP‐dependent pathways. Such time‐dependent acquisition of FSK responsiveness and serum stimulation of ALP expression only in mature osteoblasts indicate the possible presence of differentiation switches that impart competency for a subset of osteoblast developmental events that require complete serum for maximal expression. © 1994 Wiley‐Liss, Inc.
Noradrenergic modulation of albumin expression in growth‐stimulated adult rat hepatocytes in primary culture
Authors: Fabregat Isabel , De Juan Carmen , Roncero CÉSar , Benito Manuel
Published: February 2005

Serum albumin is the most abundant protein synthesized by liver cells, and its production is a reliable indicator of the differentiated state of hepatocytes. We have recently shown that fetal rat hepatocytes cultured under proliferative conditions, i.e., in the presence of EGF, responded to glucagon and noradrenaline increasing albumin protein and mRNA levels (de Juan et al., 1992,. J. Cell. Physiol., 152:95–101). This effect was mimicked by agents that increase cyclic AMP levels. In this report, we show that in regenerating liver, noradrenaline modulation of albumin expression seems to be different. Hepatocytes from hepatectomized rats were cultured at low cell density and in the presence of EGF. Under these conditions, noradrenaline, which acted synergistically with EGF increasing DNA synthesis (de Juan et al., 1992. Exp. Cell. Res., 202:495–500), produced a decrease in albumin mRNA levels. This effect was dose‐dependent, being maximum at 1 μM noradrenaline. Noradrenergic effect seemed to be mediated by α‐receptors, because it was blocked by prazosin, but not by propranolol. Other Ca2+‐increasing agents, as vasopressin, angiotensin II, or ATP, did not produce any effect. However, albumin mRNA levels decreased when the cells were incubated in the presence of tetradecanoyl phorbol‐13‐acetate (TPA). In addition, noradrenergic modulation of albumin expression was blocked by staurosporine, a protein kinase inhibitor with relative specificity for protein kinase C. Thus we can conclude that the role of noradrenaline on the regulation of liver growth and differentiation changes from fetal to adult life. This change is probably due to its action on different receptors: β‐receptors in fetal hepatocytes and α1‐receptors in the adult liver. © 1994 Wiley‐Liss, Inc.
C‐terminal fragments of parathyroid hormone‐related protein, PTHrP‐(107‐111) and (107‐139), and the N‐terminal PTHrP‐(1‐40) fragment stimulate membrane‐associated protein kinase C activity in rat spleen lymphocytes
Authors: Whitfield James F. , Isaacs Richard J. , Chakravarthy Balu R. , Durkin Jon P. , Morley Paul , Neugebauer Witold , Williams Ross E. , Willick Gordon , Rixon Raymond H.
Published: February 2005

Membrane‐associated protein kinase C (PKC) activity in lymphocytes freshly isolated from rat spleen was stimulated by the C‐terminal parathyroid hormonerelated protein fragments, PTHrP‐(107–111) and PTHrP‐(107–139), at concentrations from 10−3 to 104 pM. By contrast, the same concentrations of PTHrP‐(120–139), Without the 107–111 TRSAW (‐Thr‐Arg‐Ser‐Ala‐Trp‐) sequence of the other C terminal fragments, did not stimulate spleen lymphocyte PKC. Low concentrations of the N‐terminal PTHrP‐(1–40) fragment also stimulated membrane‐associated PKC activity in the spleen lymphocytes. These results suggest that PTHrP might be an important physiological regulator of the immune response. Published 1994 Wiley‐Liss, Inc.
Pinocytic stimulation in Dictyostelium discoideum by γ‐benzene hexachloride
Authors: Gayatri R. , Chatterjee S.
Published: February 2005

Pinocytic activity is greatly stimulated in γ‐BHC (gamma isomer of benzene hexachloride) treated, vegetative cells of Dictyostelium discoideum as measured by 14C sucrose or FITC‐dextran uptake. Transmission electron microscopic studies also reveal the presence of a greater number of pinosomal vesicles in the pesticide‐treated Dictyostelium amoebae. The enhanced pinocytic activity has been discussed in relation to lipophilic interactions of γ‐BHC with the hydrophobic cell surface and the observed changes in the cytoskeletal proteins of the treated cells. © 1994 Wiley‐Liss, Inc.