bluuebunny/crossref_metadata_2025 huggingface dataset:
TGF‐β regulation of nuclear proto‐oncogenes and TGF‐β gene expression in normal human osteoblast‐like cells
Authors: Subramaniam M. , Oursler M. J. , Rasmussen K. , Riggs B. L. , Spelsberg T. C.
Published: May 2005

Transforming growth factor‐β (TGF‐β) is present in high levels in bone and plays an important role in osteoblast growth and differentiation. In order to dissect the molecular mechanisms of action of TGF‐β on osteoblasts, the effects of TGF‐β on the steady state mRNA levels of c‐fos, c‐jun, and jun‐B proto‐oncogenes on normal human osteoblast‐like cells (hOB) and a transformed human osteoblast cell line (MG‐63) were measured. Treatment of hOBs with 2 ng/ml of TGF‐β1 resulted in a rapid increase in c‐fos mRNA levels as early as 15 min post‐treatment. A maximum (10‐fold) increase was observed at 30 min after TGF‐β treatment followed by a decrease to control values. Similar responses were measured whether the cells were rapidly proliferating or quiescent. TGF‐β1 induced jun‐B mRNA levels more gradually with steady increase initially observed at 30 min and a maximum induction measured at 2 h post‐TGF‐β treatment. In contrast, TGF‐β treatment caused a time dependent decrease in the c‐jun mRNA levels, an opposite pattern to that of jun‐B mRNA. Treatment of hOBs with TGF‐β1 in the presence of actinomycin‐D abolished TGF‐β1 induction of c‐fos mRNA, suggesting that TGF‐β action is mediated via transcription. In the presence of cycloheximide, TGF‐β causes super‐induction of c‐fos mRNA at 30 min, indicating that the c‐fos expression by TGF‐β is independent of new protein synthesis. Further, transfection of 3 kb upstream region of jun‐B promoter linked to a CAT reporter gene into ROS 17/2.8 cells was sufficient to be regulated by TGF‐β1. Interestingly, TGF‐β treatment also increased the mRNA levels of TGF‐β1 itself at 4 h post TGF‐β treatment, with a maximum increase observed at 14 h of treatment. TGF‐β1 treatment for 30 min were sufficient to cause a delayed increase in TGF‐β protein secretion within 24 h. These data support that TGF‐β has major effects on hOB cell proto‐oncogene expression and that the nuclear proto‐oncogenes respond as rapid, early genes in a cascade model of hormone action.
Sulfate restriction induces hyposecretion of the adhesion proteoglycan and cell hypomotility associated with increased 35SO42− uptake and expression of a band 3 like protein in the marine sponge, Microciona prolifera
Authors: Kuhns William J. , Popescu Octavian , Burger Max M. , Misevic Gradimir
Published: May 2005

Sulfate is an important component relating to normal proteoglycan secretion and normal motility in the marine sponge, Microciona prolifera. The following alterations were observed in sponge cells when sulfate free artificial sea water was used as the suspension medium: (1) impairment of aggregation, (2) loss of cell movements, (3) a marked reduction in the secretion of the adhesion proteoglycan (AP). Reversal of this effect occurred if sulfate depleted cells were again rotated in sulfate containing artificial sea water. Motility and reaggregation of sulfate deprived cells could be completely restored by purified AP, but only if cells were first pre‐conditioned in normal sea water. Comparisons of 35SO42− uptake between normal and sulfate deprived cells which had been treated to reduce preformed secretions showed a marked increase in 35SO42− uptake and incorporation which could be greatly augmented in the presence of Ca2+/Mg2+. Excessive retention of AP in sulfate starved cells demonstrated by immunostaining suggested that AP secretion and cellular motility may be controlled by a sulfate dependent secretogogue or that undersulfated AP itself had developed a secretory defect. SDS‐PAGE of Triton treated cellular extracts demonstrated a 116 kDa 35SO42− sulfated band which co‐migrated with AP, but only in extracts derived from sulfate starved cells. Western blots prepared from such extracts incubated in the presence of a monoclonal anti‐band 3 antibody demonstrated labelling of a single 97 kDa band only in material from sulfate deprived cells. The absence of this component in normal cell extracts indicated that this protein may be involved in facilitated sulfate transport. This study lends support to a heretofore unrecognized role for sulfate in cell motility and secretion.
Monoclonal antibody recognizing a determinant on type 2 chain blood group A and B oligosaccharides detects oncodevelopmental changes in azoxymethane‐induced rat colon tumors and human colon cancer cell lines
Authors: Laferté Suzanne , Prokopishyn Nicole L. , Moyana Terence , Bird Ranjana P.
Published: May 2005

Altered expression of ABH blood group substances is a common feature of human colorectal carcinoma, yet it remains unclear how these structural changes influence the biological properties of tumor cells. Azoxymethane‐induced rat colon tumors display many features of the human disease, thereby providing a potentially useful model to study the role of blood group substances in colon cancer progression. We have prepared monoclonal antibodies to a microsomal fraction isolated from an azoxymethane‐induced rat colon tumor and selected an antibody that detects cancer‐associated changes. Monoclonal antibody (mAb) 3A7 recognizes a determinant on type 2 chain blood group A (GalNAcα1–3[Fucα1–2]Galβ1–4GlcNAc‐R) and B (Galα1–3[Fucα1–2]Galβ1–4GlcNAc‐R) oligosaccharides. Expression of the epitope detected by this antibody was developmentally regulated in rat colon, with maximal expression from day 4–21 after birth. Immunohistochemical staining and Western blotting analyses of azoxymethane‐induced colon tumors revealed increased expression of the epitope in all of the 21 colonic tumors examined, including preneoplastic glands within transitional mucosa. Conventional and signet‐ring adenocarcinomas that had invaded through the muscularis propria (Duke's B2) consistently showed the most intense staining with mAb 3A7, including regions depicting angioinvasion. Some of the lymph node metastases (Duke's C2) stained poorly with the antibody. The epitope was also expressed in blood group A positive human colon carcinoma cell lines, including HT29 and SW480 but not by SW620, a cell line derived from a lymph node metastasis isolated in vivo from the SW480 primary tumor, or in the blood group B cell line SW1417. The glycoproteins detected by mAb 3A7 in rat colon tumors and HT29 cells ranged in size between 50 and 200 kd, including a major species of 140 kd. Affinity chromatography of detergent lysates of normal rat colon on the blood group A specific lectin Dolichos biflorus (DBA)‐agarose resulted in nearly quantitative binding of glycoprotein species detected by the antibody. By contrast, immunoreactive glycoproteins from rat colon tumors or HT29 cells bound poorly to DBA‐agarose but were retained by another blood group A‐binding lectin, Helix‐pomatia (HPA)‐agarose. These results indicate that colon carcinogenesis results in quantitative as well as qualitative changes in oligosaccharides detected by mAb 3A7 and suggest that the combined use of mAb 3A7 and blood group A‐specific lectins may provide a useful tool for early detection of colon cancer.
Inhibition of endothelial cell proliferation by SPARC is mediated through a Ca2+‐binding EF‐hand sequence
Authors: Sage E. Helene , Bassuk James A. , Yost Jeffrey C. , Folkman M. Judah , Lane Timothy F.
Published: May 2005

SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin and BM‐40) is a metal‐binding glycoprotein secreted by a variety of cultured cells and characteristic of tissues undergoing morphogenesis, remodeling, and repair. Recently it has been shown that SPARC inhibits the progression of the endothelial cell cycle in mid‐G1, and that a synthetic peptide (amino acids 54–73 of secreted murine SPARC, peptide 2.1) from a cationic, disulfide‐bonded region was in part responsible for the growth‐suppressing activity [Funk and Sage (1991): Proc Natl Acad Sci USA 88:2648–2652]. Moreover, SPARC was shown to interact directly with bovine aortic endothelial (BAE) cells through a C‐terminal EF‐hand sequence comprising a high‐affinity Ca2+‐binding site of SPARC and represented by a synthetic peptide (amino acids 254–273) termed 4.2 [Yost and Sage (1993): J Biol Chem 268:25790–25796]. In this study we show that peptide 4.2 is a more potent inhibitor of DNA synthesis that acts cooperatively with peptide 2.1 to diminish the incorporation of [3H]‐thymidine by both BAE and bovine capillary endothelial (BCE) cells. At concentrations of 0.019–0.26 mM peptide 4.2, thymidine incorporation by BAE cells was decreased incrementally, relative to control values, from approximately 100 to 10%. Although somewhat less responsive, BCE cells exhibited a dose‐responsive decrement in thymidine incorporation, with a maximal inhibition of 55% at 0.39 mM. The inhibitory effect of peptide 4.2 was essentially independent of heparin and basic fibroblast growth factor and was blocked by anti‐SPARC peptide 4.2 IgG, but not by antibodies specific for other domains of SPARC. To identify residues that were necessary for inhibition of DNA synthesis, we introduced single amino acid substitutions into synthetic peptide 4.2 and tested their activities and cell‐surface binding characteristics on endothelial cells. Two peptides displayed null to diminished effects in the bioassays that were concentration‐dependent: peptide 4.2 K, containing an Asp258 → Lys substitution, and peptide 4.2 AA, in which the two disulfide‐bonded Cys (positions 255 and 271) were changed to Ala residues. Peptide 4.2 K, which failed to fulfill the EF‐hand consensus formula, exhibited an anomalous fluorescence emission spectrum, in comparison with the wild‐type 4.2 sequence, that was indicative of a compromised affinity for Ca2+. Moreover, ablation of the disulfide bond in peptide 4.2 AA potentially destabilized the Ca2+‐binding loop structure, as assessed by fluorescence spectroscopy, such that the peptide competed poorly for the binding of [125I]‐peptide 4.2 to BAE cells. We conclude both that Ca2+‐coordinating Asp at position 258 and the conformation of peptide 4.2 are necessary for the inhibition of DNA synthesis by SPARC in cultured endothelial cells.
Integrity of intermediate filaments is associated with the development of acquired thermotolerance in 9L rat brain tumor cells
Authors: Lee Yu‐Chien , Lai Yiu‐Kay
Published: May 2005

Withangulatin A (WA), a newly discovered withanolide isolated from an antitumor Chinese herb, has been shown to be a vimentin intermediate filament‐targeting drug by using immunofluorescence microscopy. Together with cytochalasin D and colchicine, these drugs were employed to investigate the importance of vimentin intermediate filaments, actin filaments, and microtubules in the development of acquired thermotolerance in 9L rat brain tumor cells treated at 45°C for 15 min (priming heat‐shock). Acquired thermotolerance was abrogated in cells incubated with WA before the priming heat‐shock but it could be detected in cells treated with WA after the priming heat‐shock. In contrast, cytochalasin D and colchicine do not interfere with the development of thermotolerance at all. The intracellular localizations of vimentin and the constitutive heat‐shock protein70 (HSC70) in treated cells were examined by using immunofluorescence microscopy and detergent‐extractability studies. In cells treated with WA before the priming heat‐shock, vimentin IFs were tightly aggregated around the nucleus and unable to return to their normal organization after a recovery under normal growing conditions. In contrast, the IF network in cells treated with WA after the priming heat‐shock was able to reorganize into filamentous form after a recovery period, a behavior similar to that of the cells treated with heat‐shock only. HSC70 was found to be co‐localized with vimentin during these changes. It is suggested that the integrity of intermediate filaments is important for the development of thermotolerance and that HSC70 may be involved in this process by stabilizing the intermediate filaments through direct or indirect binding.
Differentiation in human endometrial cells in monolayer culture: Dependence on a factor in fetal bovine serum
Authors: Fleming Honorée
Published: May 2005

Human epithelial cells of the Ishikawa endometrial line can be stimulated to differentiate and form multicellular structures in 4–5 day‐old monolayer cultures by the addition of a protein factor from fetal bovine serum. Multicellular structures become obvious over an 18–30‐h period as the cells enlarge, separate from the dish, and form domes. These structures are similar to those that result from polarization in other epithelial cell lines. Ishikawa dome formation appears to be a multistage process. The appearance of enlarged differentiated cells is detected within hours of adding fetal bovine serum; these enlarged cells lift off the surface of the dish within 6–8 more hours. Domes are observed about 24 h after the addition of fetal bovine serum. Sometimes dome cells migrate into a “bud‐like” structure that extends out from the dome. Differentiation of the domes is dependent on a factor from fetal calf serum that behaves similarly to a very large protein or complex of proteins, greater than 300 kd. Progesterone appears to enhance the formation of domes but does not elicit dome formation in the absence of serum factor.
Prolactin induced expression of interleukin‐1 alpha, tumor necrosis factor‐alpha, and transforming growth factor‐alpha in cultured astrocytes
Authors: DeVito William J. , Avakian Crystal , Stone Scot , Okulicz William C. , Tang Kam‐Tsun , Shamgochian Maureen
Published: May 2005

Prolactin (PRL) is a potent mitogen in cultured astrocytes. Because one of the major effects of astrocyte proliferation is the expression of inflammatory cytokines, we examined the effect of PRL‐induced mitogenesis on the expression of interleukin‐1 (IL‐1α), tumor necrosis factor‐α (TNF‐α), and transforming growth factor‐α (TGF‐α) in cultured astrocytes. Astrocytes were stimulated with PRL or growth hormone (GH), and the expression of cytokines was determined by immunohistochemistry and Western blot analysis. Following incubation of astrocytes with 1 nM PRL for 6 h, strong positive staining of IL‐1α and TNF‐α, but not TGF‐α, was found. No detectable staining for the above cytokines was found in vehicle, or GH treated astrocytes. When astrocytes were incubated in the presence of 1 nM PRL for 18 h, strong positive staining for IL‐1α and TGF‐α was found. Immunocytochemical analysis of the expression of TNF‐α and IL‐1α in PRL stimulated astrocytes suggested that the expression of IL‐1α preceded the expression of TNF‐α. To confirm this observation, Western blot analyses were performed on extracts from astrocytes incubated with 1 nM PRL. In unstimulated astrocytes, IL‐1α levels were not detectable. In astrocytes stimulated with 1 nM PRL, expression of IL‐1α was clearly detected after 1 h of incubation, and IL‐1α levels continued to increase during the course of the experiment (6 h). In contrast, in astrocytes stimulated with 1 nM PRL, an increase in the expression of TNF‐α was first apparent after 2 h of incubation. TNF‐α levels peaked 3 to 4 h after the addition of PRL, and returned to near control levels after 6 h. Finally, injection of PRL into a wound site in female rats increased the expression of glial fibrillary acid protein (GFAP), an astrocyte specific protein. These data suggest that PRL can stimulate astrogliosis at the wound site in vivo. These data clearly indicate that PRL can stimulate the expression of TNF‐α and IL‐1α in cultured astrocytes and suggest that PRL may play a role in the regulation of the neuroimmune response in vivo.
Isolation of a cDNA encoding a growth‐arrest associated gene and characterization of its regulation
Authors: Ge Lynn , Liau Gene
Published: May 2005

We are interested in understanding the molecular events associated with the growth‐arrest of vascular SMCs. We constructed a subtracted cDNA library enriched in nucleotide sequences associated with quiescent SMCs. This library was screened with similarly subtracted 32P‐labeled cDNAs to identify growth‐arrest associated cDNA clones. Characterization of 19 of these cDNA clones revealed that 9 hybridized to mRNAs that exhibited a 2–3 fold increase in growth‐arrested SMCs. In addition, two other cDNAs hybridized to a 5 Kb mRNA that was elevated approximately 10‐fold in high density growth‐arrested SMCs. Genomic Southern blot hybridization and DNA sequencing analysis indicated that these cDNAs encoded the same gene (LG7) and that this gene may be a member of a multigene family or that it may contain a sequence shared by other unrelated genes. Augmented expression of LG7 was associated with both high cell density and serum deprivation induced growth‐arrest. LG7 mRNA expression was down‐regulated when SMCs were incubated with FBS or with reagents that arrest cells in early S‐phase. Additional analysis with cell cycle specific inhibitors indicated that LG7 mRNA levels were also low when cells were blocked at the G2 phase of the cell cycle but blockage at mitosis resulted in an elevated level of LG7 mRNA. We further demonstrated that the expression of LG7 was dependent on the presence of a relatively labile protein since protein synthesis inhibitors specifically blocked the expression of this mRNA but not the mRNA expression of α1(III) collagen or ferritin H‐chain. Finally, we demonstrated that Bt2cAMP was able to induce mRNA expression of LG7 within 2 h, suggesting that this gene may be directly regulated via the cyclic‐AMP‐dependent protein kinase pathway.
Inhibition of extracellular ATP‐mediated lysis of human macrophages by calmodulin antagonists
Authors: Blanchard D. Kay , Hoffman Susan L. , Djeu Julie Y.
Published: May 2005

Lysis of human culture‐derived macrophages by extracellular ATP has recently been described, and treatment of macrophages with interferon‐γ rendered those cells significantly more sensitive to lysis. In addition, cell death occurred more rapidly in interferon (IFN)‐treated cells than in untreated macrophages. In an attempt to identify the mechanism by which extracellular ATP affects macrophages, as well as to explore the differences between interferon‐γ‐treated and untreated macrophages, selected metabolic inhibitors were included in the lytic assays. Of the compounds tested, three antagonists of calmodulin‐linked pathways (trifluoperazine, KN‐62, and calmidazolium) blocked the ATP‐mediated lysis of both interferon‐γ‐treated and colony‐stimulating factor‐treated macrophages in a dose‐dependent manner. Early signals of the ATP ligation of the P2Z purinoceptors of human macrophages included increases in cytosolic [Ca2+] and depolarization of the plasma membrane. However, the inclusion of calmodulin antagonists in these assays did not abrogate either effect. These results suggest that the mechanism which mediates the efflux of 51Cr‐labeled proteins from ATP‐lysed macrophages is distinct from calcium mobilization and membrane depolarization, and may involve the generation of secondary pores channels in the plasma membrane via a calmodulin‐linked pathway.
In vivo protein‐DNA interactions at the c‐jun promoter in quiescent and serum‐stimulated fibroblasts
Authors: Rozek Dieter , Pfeifer Gerd P.
Published: May 2005

c‐Jun is an important component in the regulation of cell proliferation. As a member of the early response gene family, c‐jun is induced within minutes in the presence of mitogenic agents such as serum growth factors. Using in vivo footprinting, we have analyzed protein‐DNA interactions at the c‐jun promoter in human fibroblasts subjected to growth arrest and serum stimulation. We located seven footprints upstream of the transcription initiation site. Protein‐DNA interactions were detected at two AP‐1–like sequences, a CCAAT box, an SP‐1 sequence, an NF‐jun sequence, a putative RSRF (related to serum response factor) binding site, and a sequence bound by an unknown factor. All of these binding sites were occupied in serum‐starved cells, and no additional protein‐DNA interactions were detected upon serum stimulation. Evidence from this study supports a model in which expression of the c‐jun gene is mediated by phosphorylation events taking place on the transactivation domains of promoter‐bound transcriptional activators.
EGF and TGFα modulate structural and functional differentiation of the mammary gland from pregnant mice in vitro: Possible role of the arachidonic acid pathway
Authors: Spitzer Eva , Zschiesche Wolfgang , Binas Bert , Grosse Richard , Erdmann Bettina
Published: May 2005

Epidermal growth factor (EGF) has been suggested to be involved in mammary gland development by mitogenic stimulation of the ductal and alveolar epithelium in virgin mice. The present studies demonstrate that also in late‐pregnant mice EGF leads to proliferation of the ductal, ductular, and alveolar epithelium. The mitogenic effect is associated with structural and functional dedifferentiation of alveolar cells as revealed by analysis of morphology, expression of cytosolic and secretory proteins, and fatty acid synthesis. Using a combination of metabolic inhibitors, the dedifferentiating effect of EGF could be blocked while the mitogenic action was not influenced. This finding demonstrates that the signal transduction pathway leading to dedifferentiation and mitosis can be separated, and that the dedifferentiating effect of EGF is independent of its mitogenic properties, but is probably mediated by activation of the arachidonic acid‐dependent pathways (cyclo‐ and lipoxygenase pathways). Release of arachidonic acid from the endogenous phospholipid pool was found to be an early response of the explants to EGF. Accordingly, arachidonic acid itself proved to be capable of inducing epithelial dedifferentiation but failed to stimulate proliferation. TGFα showed qualitatively similar effects as EGF but was generally a stronger agonist. It is suggested that EGF and TGFα also play a role in mammary gland physiology during pregnancy by final developing and maintanance of the lobulo‐alveolar structure in the mammary gland and prevention of premature onset of lactation, and that this is mediated through the PLA2‐arachidonic acid signalling cascade.
Glucocorticoid amplifies vasopressin‐induced phosphoinositide hydrolysis in aortic smooth muscle cells
Authors: Watanabe Yasuko , Tokuda Haruhiko , Suzuki Atsushi , Shinoda Junji , Kotoyori Jun , Ito Yoshiaki , Oiso Yutaka , Kozawa Osamu
Published: May 2005

It has been reported that glucocorticoid modifies phosphoinositide (PI) hydrolysis stimulated by vasoactive agents in vascular smooth muscle cells. In the present study, we investigated the point at which glucocorticoid affects vasopressin‐induced PI hydrolysis in primary cultured rat aortic smooth muscle cells. The pretreatment with dexamethasone significantly amplified the formation of inositol trisphosphate (IP3) induced by vasopressin in a dose‐dependent manner in a range of 1 pM to 10 nM. The effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone had little effect on the number of vasopressin receptor and its affinity to vasopressin. The pretreatment with dexamethasone also amplified the formation of IP3 induced by NaF, a GTP‐binding protein activator, or angiotensin II. 12‐O‐Tetradecanoylphorbol‐13‐acetate, a protein kinase C (PKC)‐activating phorbol ester, significantly reduced the dexamethasone‐induced enhancement of IP3 formation stimulated by vasopressin, angiotensin II or NaF. 4α‐Phorbol‐12, 13‐didecanoate, a PKC‐nonactivating phorbol ester, had little effect on the enhancement by dexamethasone. These results strongly suggest that glucocorticoid amplifies vasopressin‐induced PI hydrolysis at a point downstream from GTP‐binding protein in primary cultured rat aortic smooth muscle cells, and that the activation of PKC has a negative feedback effect on the amplification by glucocorticoid of vasopressin‐induced PI hydrolysis.
Activation of the C‐fos promoter by increased internal pH
Authors: Murguía José R. , De Vries Luc , Gomez‐García Lourdes , Schönthal Axel , Perona Rosario
Published: May 2005

Changes in intracellular pH (pHin) take part in the mitogenic response. Their importance has been stressed by the finding that mouse fibroblasts expressing a yeast proton pumping ATPase (PMA1) exhibit a transformed phenotype and are tumorigenic. These cells do maintain a higher pHin, supporting the idea that elevated pHin may act as a proliferative trigger. Here we show that cells constitutively expressing PMA1 have higher levels of the AP‐1 transcription factor. The use of stable transfectants and transient transfection assays show that PMA1 activity induces transactivation of the c‐fos promoter. The activation of the promoter is mediated throughout the serum response element (SRE). The use of protein kinase C inhibitors suggests that AP‐1 activation is achieved through a pathway independent of protein kinase C.
Estimation of PCNA mRNA Stability in cell cycle by a serum‐deprivation method
Authors: Liu Yin‐Chang , Chen Gung‐Shen , Liu Wei‐Li , Wen Su‐Fang
Published: May 2005

A simple scheme was developed to study the mRNA stability of the proliferating cell nuclear antigen (PCNA) gene during cellular transition from the G1/S boundary to a quiescent state. By this scheme, CHO.K1 cells were grown to about 80% confluence and then serum‐starved for 40 h for synchronization in a quiescent state. The quiescent cells were serum‐stimulated for a period of time (between 8 h and 12 h) and then grown in serum‐free medium until being harvested for further analyses. The cellular PCNA mRNA level was analyzed by Northern blotting. As compared with that in cells which were continuously incubated in serum‐containing medium, the decline of the mRNA level, after reaching the peak, in these serum‐deprived cells was virtually devoid of mRNA synthesis. Thus, this mRNA decay was taken for the measurement of mRNA stability. The advantage of the scheme is that, unlike the treatment of transcription inhibitors, it does not prevent the cells from completing the rest of the cell cycle before returning to the resting state, and so the mRNA stability observed is cell cycle dependent. In contrast with the previous report that the stability of PCNA mRNA in quiescent cells is less by severalford than that in S phase cells, our study shows that the mRNA stability of PCNA remained constant during the cellular transition from G1/S boundary to quiescent state.
Heritable formation of neuroectodermal tumor in transgenic mice carrying the combined e1 region gene of adenovirus type 12 with the deregulated human renin promoter
Authors: Sugiyama Fumihiro , Sagara Masashi , Matsuda Yoichi , Horiguchi Hisashi , Kamma Hiroshi , Ogata Takesaburo , Hatae Toshihisa , Yagami Ken‐Ichi , Murakami Kazuo , Fukamizu Akiyoshi
Published: May 2005

Adenovirus early 1 (E1) region gene products, including E1A and E1B, are required for transcriptional regulation of viral and cellular promoters in infected and transfected culture cells and for transformation of primary rodent cells. Here, we established a line of transgenic mice carrying the E1 region gene of human adenovirus type 12 under the control of the human renin promoter, in which a neuroectodermal tumor derived from retroperitoneal, olfactory, and/or pelvic regions was heritably developed with varying degrees of incidence and the phenotype was successfully passed through six generations. The transgenes were located in the region E2‐E3 bands of chromosome 7 with which no genetic linkage to neuroectodermal tumors was previously demonstrated, and expressed only in the tumors but not in another tissue examined. Notably, in addition to the expression of a neural marker gene N‐CAM, the three nuclear oncogenes, c‐, L‐, and N‐myc, were coexpressed in the tumors. These results suggest that E1A and E1B are cooperatively involved in the heritable formation of neuroectodermal tumors associated with co‐expression of the three sets of myc family genes.
Sequence‐specific DNA binding activities of nuclear matrix proteins of mammalian lens epithelial cells
Authors: Bagchi M. , Van Wijnen A. , Katar M. , Merriman H. , Lian J. , Stein J. , Stein G. , Maisel H.
Published: May 2005

This study examines matrix and nonmatrix nuclear proteins of the rabbit lens epithelial cells. The nuclear matrix proteins were isolated by modified Penman technique, which requires presence of detergents and nucleases, whereas nonmatrix nuclear proteins were obtained by high salt extraction. The data from these experiments revealed presence of DNA binding activities for SP‐1 and OCT‐1 proteins in both matrix and non‐matrix compartments of rabbit lens epithelial cells. Comparison of the relative abundance of SP‐1 and OCT‐1 binding activities in nuclear matrix and nonmatrix fraction suggest the distribution between these two compartment is cell type specific and possibly related to the control of cell growth. © Wiley‐Liss, Inc.
Acidic and basic fibroblast growth factor bind with differing affinity to the same heparan sulfate proteoglycan on BALB/c 3T3 cells: Implications for potentiation of growth factor action by heparin
Authors: Brown Kathryn J. , Hendry Ian A. , Parish Christopher R.
Published: May 2005

Heparan sulfate proteoglycans on the cell surface act as low affinity binding sites for acidic and basic fibroblast growth factor (FGF) [Moscatelli (1887): J Cell Physiol 131:123–130] and play an important role in the interaction of FGF with the FGF receptor (FGFR). In this study, several aspects of the interaction of FGFs with cell surface heparan sulfate proteoglycans were examined. Reciprocal cross blocking studies demonstrated that acidic FGF (aFGF) and basic FGF (bFGF) bind to identical or closely associated heparan sulfate motifs on BALB/c 3T3 cell surface heparan sulfate proteoglycans. However, the binding affinity of the two growth factros for these heparan sulfate proteoglycans differs considerably, competition binding data indicating that aFGF has a 4.7‐fold lower affinity than bFGF for 3T3 heparan sulfate proteoglycan. Subsequent studies of dissociation kinetics demonstrated that bFGF dissociates form the FGFR at least 10‐fold slower than aFGF, whereas, following removal of cell surface heparan sulfate proteoplycan. Subsequent studies of dissociation kinetic demonstrated that bFGF dissociates from the FGFR at least 10‐fold slwer than aFGF, whereas, following removal of cell surface heparan sulfate proteoglycans by heparinase treatment, the dissociation rate of both FGFs is similar and rapid. These results support the concept that cell surface heparan sulfate proteoglycans stabilize the interactio fo FGF with FGFR, possibly by the formatin of a ternary complex. © Wiley‐Liss, Inc.
Myoblast and myotude nuclei display similar patterns of heterogneous acetylcholine receptor subunit mRNA expression
Authors: Su Xing , Berman Stephen A. , Sullivan Thomas , Bursztajn Sherry
Published: May 2005

Muscle progenitor cells differentiate to myoblasts, and subsequently myotubes, upon expression of muscle specific genes. We and others have previously shown that myotude nuclei, even in the absence of nerve, express AChR α subunit RNA at varying levels, with a small subset (about ten percent) of the nuclei expressing at high levels. These findings raised two important quwstions:(1) is the observed heterogeneneity a unique property of the α subunits, and (2) when does the heterogeneneity begin? In particular, is it induced only at or afer the time of fusion, or does it exist at the myoblast stage? We hasve, therfore, extended our observations to the γ and δ subunits and we also have examined the distributions of AChR α,γ, and δ subunit RNAs in both myoblasts and myotubes. We used intron and intron‐exon probes to detect prespliced trascripts or mature mRNAs in the cells. Because inton‐containing transcripts are not transported out of the nuclei, the distributions of these transcripts can indicate their expression patterns among nuclei in the same myotubes. Our results show that both myotubes and myotubes have distribution of the AChR α,γ, and δ subunit RNAs which differ sharply from that of the U1 RNA or Myo D. Thus, the heterogeneous expression of AChR genes is not only an intrinsic property of muscle cell nuclei (in the sense that it does not require the presence of nerves), but it also exists prior to fusion. Our results suggest that muscle nuclei attain individualized capacities for AChR subunit mRNA production early in their development. Conceptual models consistend with individuality imply an additional level of regulation beyond the known diffusible transcriptional factors. © Wiley‐Liss, Inc.
Intranuclear binding on nucleoplasmin
Authors: Paine Philip L. , Yassin Rihab , Paine Terry Maloney , Vancurva Ivana , George Cathleen , Sahay Neayka
Published: May 2005

Many proteins—including not only structural proteins, but also enzymes, hormone receptors, and other transcription factors—accumulate to much higher nuclear than cytoplasmic concentrations. Nuclear Localization sequences or signals (NLSs) within their primary structures entrain specific transport of these proteins through the nuclear pore complexes. Thios transport process is energy‐dependent, but evidence for a true active transport mechanism is not conclusive. An alternative mechanism—facilitated transport of NLS proteins followed by their intranuclear binding—has been implicated by experiments with oil‐isolated nuclei. However, there has been no agreement as to a role for binding in the in vivo nuclear accumulation of NLS‐containing proteins. We Demonstrate herein that a prototypical NLs protein, Nucleoplasmin (NP), binds within the nucleus of the living Xenopus oocyte and that this binding accounts for its nuclear accumulation. © Wiley‐Liss, Inc.
5′ Phosphorylation of DNA in mammalian cells: Identification of a polymin P‐precipitable polynucleotide kinase
Authors: Prinos Panagiotis , Slack Carolyn , Lasko Dana D.
Published: May 2005

Proteins that catayze 5′ phosphorylation of an oligodeozyribonucleotide substrate can be fractionated by polumin P treatment of whole cell extrats of calf thymus glands. Anion exchange chromatography on Q‐Sepharose revealed three separable peaks of activity in the polymin P supernatant fraction, and one peak of activity in the Polymin P pellet fraction. The latter activity, polymin P‐precipitable polynucleotide kinase (PP‐PNK), was futher purified with a 1,500‐fold increase of specific activity compared to the crude polymin fraction. Oligonucleotides, a dephosphorylated 2.9‐kb EcoRI fragment, and poly(A) were phosphorylated by the enzyme preparation, but thymidine 3′monophosphate was not a substrate. PP‐PNk preparations exhibited an apparent KM of 52 μM for ATP and 8 μM for oligo dT25. The enzyme preparation displayed no detectable 3′ phosphatase or cyclic 2′,3′ phosphohydrolase activities. The sedimentation coefficient of the PP‐Pnk activity was 3.85 as determined by sucrose density gradient analysis; the stokes radius was 45 Å, leading to an estimated molecular mass of 72 kDa. The enzyme had a pH optimun in the neutral to alkaline range in several buffer systems and is distinct from the DNA Kinase with an acidic pH optimum previously described in calf thymus. © Wiley‐Liss, Inc.
Cells en route to apoptosis are characterized by the upregulation of c‐fos, c‐myc, c‐jun, cdc2, and RB phosphorylation, resembling events of early cell‐cycle traverse
Authors: Pandey Siyaram , Wang Eugenia
Published: May 2005

Density‐arrested quiescent murine Balb/c‐3T3 cells are dependent upon growth factors for their survival. Withdrawal of serum from their medium induces rapid cell death, the mechanism of which is not yet fully understood. We have studied the effect of serum deprivation on density‐inhibited quiescent Swiss 3T3 cells and found that they undergo rapid cell death upon total withdrawal of serum. The nature of this cell death is similar to apoptosis, as shown by cellular and nulear morphology and DNA fragmentation into oligonucleosomal fragments. Investigating the regulation of early cell‐cycle genes during this process, we found that c‐myc, c‐jun, c‐fos, and cdc2, protein presence is induced after serum deprivation, when the phosphorylated form of the RB protein also appears. The upregulation of these genes' protein products is coupled with the appearance of PCNA, a proliferation‐specific nuclear antigen, as well as significant incorporation of BrdU, which may reflect DNA repair activity; in situ analysis shows that BrdU‐positive cells are also positive for DNA fragmentation. These results suggest that en route to apoptosis, cells undergo events typical of early cell‐cycle traverse by expressing early G1 genes and may even experience the late G1/S phase boundary, as shown by the presence of PCNA. However, the demonstrated ability of these cells to traverse the G1 phase of the cell cycle seems to be an abortive event, since they die shortly afterwards.
Suramin inhibits C6 glioma‐induced angiogenesis in vitro
Authors: Coomber Brenda L.
Published: May 2005

Aspects of tumor‐induced angiogenesis in vitro were examined using an assay involving collagen gel invasion by a surface monolayer of bovine endothelial cells under the influence of serum free conditioned medium produced by C6 cells, an experimentally derived rat glial tumor cell line. The effects of the polyanionic compound suramin, known to interfere with growth factor/cell signaling on this process were evaluated. Collagen gel invasion was quantified by adding C6 conditioned medium with or without various doses of suramin to monolayers of bovine aortic endothelial cells grown on type I collagen gels in transwell inserts. Cultures were monitored with phase‐contrast microscopy. After various periods of incubation collagen gels were fixed, embedded in epoxy resin, and 1‐μm thick sections were stained with toluidine blue. Additional cultures were used to evaluate the effects of C6 conditioned medium and suramin on endothelial cell proliferation, and on chemotaxis through 8‐μm pores. C6 glioma cell conditioned medium induced large vessel endothelial cells to sprout into the underlying collagen matrix and subsequently from networks of capillary like tubes. Conditioned medium was also chemotactic and mitogenic for these cells. The addition of suramin to C6 glioma conditioned medium prevents tube formation in collagen gels, and inhibits both endothelial cell proliferation and chemotaxis in a dose dependent manner. These results suggest that glial tumor cell conditioned medium induces angiongenesis in large vessel endothelial cells in vitro via mechanisms which are disrupted by suramin, most likely involving tumor‐derived growth factor release and/or endothelium‐mediated matrix proteolysis.
Collagen binding activity of recombinant and N‐terminally modified annexin V (anchorin CII)
Authors: Turnay Javier , Pfannmüller Eva , Lizarbe María Antonia , Bertling Wolf M. , von der Mark Klaus
Published: May 2005

We have cloned the full coding cDNA sequence of chicken annexin V and of a mutant lacking 8 amino acid residues of the N‐terminal tail for prokaryotic expression. Both proteins were synthesized in Escherichia coli upon induction with isopropyl thio‐β‐D‐galactoside, and were purified following two different protocols: one based on the ability of these proteins to interact reversibly with liposomes in the presence of calcium, and the other based on two sequential ion‐exchange chromatographic steps. Spectroscopical analysis of recombinant annexin V revealed that binding of calcium did not change the circular dichroism spectra indicating no significant changes on the secondary structure; however, a conformational change affecting the exposition to the solvent of the tryptophan residue 187 was detected by analysis of fluorescence emission spectra. Recombinant annexin V binds with high affinity to collagen types II and X, and with lower affinity to collagen type I in a calcium‐independent manner. Heat denaturing of collagen decreases this interaction while pepsin‐treatment of collagen almost completely abolishes annexin V binding. Mutated annexin V interacts with collagen in a similar way as the nonmutated recombinant protein, indicating that the N‐terminal tail of annexin V is not essential for collagen binding.
Effects of c‐Jun and a negative dominant mutation of c‐Jun on differentiation and gene expression in lens epithelial cells
Authors: Rinaudo Jo Ann , Vacchiano Emanuel , Zelenka Peggy S.
Published: May 2005

We have used a retroviral vector (RCAS) to overexpress wild‐type chicken c‐Jun or a deletion mutant of chicken c‐Jun (JunΔ7) lacking the DNA binding region to investigate the possible role of c‐Jun in lens epithelial cell proliferation and differentiation. Both constructs were efficiently expressed in primary cultures of embryonic chicken lens epithelial cells. Overexpression of c‐Jun increased the rate of cell proliferation and greatly delayed the appearance of “lentoid bodies,” structures which contain differentiated cells expressing fiber cell markers. Excess c‐Jun expression also significantly decreased the level of βA3/A1‐crystallin mRNA, without affecting αA‐crystallin mRNA. In contrast, the mutated protein, JunΔ7, had no effect no proliferation or differentiation but markedly increased the level of αA‐crystallin mRNA in proliferating cell cultures. These results suggest that c‐Jun or Jun‐related proteins may be negative regulators of αA‐ and βA3/A1‐crystallin genes in proliferating lens cells.
Combination of insulinomimetic agents H2O2 and vanadate enhances insulin receptor mediated tyrosine phosphorylation of IRS‐1 leading to IRS‐1 association with the phosphatidylinositol 3‐kinase
Authors: Wilden Peter A. , Broadway Dale
Published: May 2005

To analyze the mechanism of action of the insulinomimetic agents H2O2, vanadate, and pervanadate (H2O2 and vanadate), CHO cells or CHO cells that overexpress wild‐type or mutant insulin receptor and/or the insulin receptor substrate (IRS‐1) were used. H2O2 or vanadate treatment alone had little or no effect on tyrosine phosphorylation of cellular proteins; however, pevanadate treatment dramatically enhanced tyrosine phosphorylation of a number of proteins including the insulin receptor and IRS‐1. However, the insulin receptor and IRS‐1 coimmunoprecipitate from insulin‐treated but not from pervanadate‐treated cells. Pervanadate‐induced tyrosine phosphorylation of the insulin receptor led to an increase in insulin receptor tyrosine kinase activity toward IRS‐1 in vivo and IRS‐1 peptides in vitro equal to that induced by insulin treatment. Pervanadate‐enhanced phosphorylation of IRS‐1 led to a fifteenfold increase in IRS‐1–associated phosphatidylinositol (Ptdlns) 3‐kinase activity. However, insulin receptor–associated Ptdlns 3‐kinase activity from pervanadate‐treated cells was not detectable, while insulin receptor–associated Ptdlns 3‐kinase activity from insulin‐treated cells was 20% of the IRS‐1‐associated activity. Thus, pervanadate but not H2O2 or vanadate alone under these conditions mimics many of insulin actions, but pervanadate treatment does not induce insulin receptor/IRS‐1 association.
U2‐SnRNP B″ protein gene is an early growth‐inducible gene
Authors: Laitinen Jens , Saris Per , Hölttä Erkki , Pettersson Ingvar
Published: May 2005

In this work we isolated mouse U2‐snRNP–specific b″ clones and analysed the expression of the mouse U2‐snRNP–specific b″ and U1‐snRNP–specific 70K genes in NIH‐3T3 fibroblasts. Stimulation of growth‐arrested NIH‐3T3 cells with serum was found to evoke a rapid increase in the amount of cytoplasmic b″ and 70K mRNAs. These increases in mRNA did not require de novo protein synthesis. Moreover, the inhibition of protein synthesis by cycloheximide caused a superinduction in the amounts of the U1‐snRNP–specific 70K transcripts. We also found that c‐Ha‐rasval12 oncogene‐transformed NIH‐3T3 cells have higher of the b″ and 70K mRNAs than the normal 3T3 cells. These data imply that the b″ and 70K are early growth response genes, and their enhanced expression might be of significance in the processing of pre‐mRNAs into mature mRNAs.
G/C element contributes to the cell line–specific expression of the proximal osteocalcin promoter
Authors: Goldberg Daniella , Gardiner Edith , Morrison Nigel A. , Eisman John A.
Published: May 2005

Sequential activation of cell type–specific genes occurs during osteoblast development. The promoter of one such gene, osteocalcin, has been widely studied, but the DNA sequences that govern osteoblast‐specific expression have not been defined. The proximal osteocalcin promoter linked to pTKCAT directs strong promoter activity in osteoblast‐like ROS17/2.8 cells and comparatively weak promoter activity in nonosteoblastic NIH3T3 cells. To identify sequences important in conferring cell‐specific expression of the osteocalcin gene, a deletion series of the human proximal promoter was constructed and the activities assessed in ROS17/2.8 and NIH3T3 cells. These studies identified a 30 bp sequence within the proximal promoter (osteocalcin repressor element‐1 [ORE‐1]) which is responsible for repressing the transcriptional activity in NIH3T3 cells. In electrophoretic mobility shift assays from both NIH3T3 and ROS17/2.8 cells, a protein complex bound to the ORE‐1 that was related to a complex which binds the G/C‐rich repressor element in the collagen type I (α1) promoter. In addition, there was a second complex from NIH3T3 cells but not ROS17/2.8 cells that bound the ORE‐1 fragment. The presence of this additional factor in NIH3T3 cells parallels the observation that constructs carrying the ORE‐1 sequence have repressed promoter activity relative to the analogous constructs lacking the ORE‐1 when transfected into NIH3T3 and suggests that the NIH3T3‐specific factor is a repressor. These data indicate that the G/C element in the ORE‐1 contributes to the repression of osteocalcin gene transcription in a nonosteoblast cell line. The high homology between the ORE‐1 sequence and a related sequence and a related sequence in the collagen type I (α2) proximal promoter suggests that homologous regions in other osteoblast‐expressed genes may function similarly.
1,25‐Dihydroxyvitamin D3: A novel agent for enhancing wound healing
Authors: Tian Xiao Q. , Chen Tai C. , Holick Michael F.
Published: May 2005

1,25‐Dihydroxyvitamin D3 (1,25(OH)2D3), has diverse effects in a variety of tissues and cell types, including skin. Since 1,25(OH)2D3 affects both fibroblast and keratinocytes, we evalauated the effect of 1,25(OH)2D3 or wound healing. We investigated the effect of the topically applied 1,25(OH)2D3 or vehicle on the healing of cutaneous wounds in rats in a blinded manner. Wound areas were measured by planimetry technique. Healing was expressed as the percentage of the original wound area that was healed. 1,25(OH)2D3 at concentrations between 5 and 50 ng/day caused a dose‐dependent acceleration of healing. Time course and specificity studies indicated that 1,25(OH)2D3 specifically promoted healing between 1–5 days after wounding as compared with vitamin D (0.5 μg/day), which showed no significant improvement over control. Our results suggest that 1,25(OH)2D3 and its analogues may be a new class of compounds that could be developed to enhance wound healing. © 1995 Wiley‐Liss, Inc.