bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractAltered 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

AbstractSPARC (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

AbstractWithangulatin 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

AbstractHuman 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

AbstractProlactin (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

AbstractWe 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

AbstractLysis 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

Abstractc‐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

AbstractEpidermal 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

AbstractIt 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

AbstractChanges 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

AbstractA 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

AbstractAdenovirus 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.