bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth‐associated gene expression is not constant in cells traversing G‐1 after exiting mitosis
Authors: Cosenza Stephen C. , Carter Ruth , Pena Angel , Donigan Anne , Borrelli Michael , Soprano Dianne Robert , Soprano Kenneth J.
Published: February 2005

Analysis of gene expression following stimulation of growth‐arrested cells has beei the main approach for identification of growth‐associated genes. Since the activation of these gene sequences is dependent on both the stimulatory agent and theitate of quiescence of the cell, the activation and role of the same genes may be entirely different in non‐growth arrested, actively proliferating cells. We have addressed the question of growth‐associated gene expression during active growth by analyzing gene expression during G‐1 of cells which have jusl exited mitosis without first leaving the cell cycle. We were able to isolate, by a non‐inductive, drug free system, a population of highly synchronized Swiss 3T3 cells within mitos is (>90%) in numbers sufficient to determine the pattern of expression pf a large number of representative growth‐associated genes. Our results show that after replating the mitotic ceils into conditioned medium: (1) growth‐associated gene expression is not constant during G‐1 of actively proliferating cells, and (2) while a number of genes (e.g., JE, c‐myc, ODC, p53, and histone) exhibited patterns of expression similar to that reported in the quiescent systems, others (e.g., nur‐77, vimentin, calcyclin) exhibited patterns which were completely different. From these results, we can begin to construct a temporal map of G‐1 progression during active growth.
Evidence for cooperativity of protein dissolution in Brij 58 permeabilized L929 cells
Authors: Ridsdale J. A. , Clegg J. S.
Published: February 2005

Mouse L929 cells were exposed to the nonionic detergent Brij 58. As has been shown in some other cell types, protein leaked from Brij 58 exposed cells only after a lag phase. In the current study we have extended the observations of the kinetics of protein efflux using cultured L cells subjected to treatment with buffers containing Brij 58. The results show that while the cells become permeable essentially at first exposure to the detergent, proteins do not scape immediately. This lag in efflux is at least partly dependent on the concentration of detergent such that a greater lag is seen in cells exposed to the lowest concentrations of Brij. Data are presented that are most readily interpreted as protein leakage having occured fairly rapidly from individual cells and that show that the time course of protein efflux results, to a large extent, from different sensitivities of individual cells to the detergent. The perrneabilized suspension cells consist of only two types, whereas the conversion of cells from one type to the other occurs through the loss of protein to the permeabilization medium. Only two bands are seen in continuous density gradients and there is a conversion of the more dense type to the less dense with longer exposure to detergent. Moreover, the less dense cells contained about half of the protein per cell as the bottom banding cells, and the proteins of the more dense cells appear to be the sum of those released into the permeabilization medium plus those found in the less dense cells.
Correlation of the inability to sustain growth in defined serum‐free medium with the suppression of tumorigenicity in Wilms' nephroblastoma
Authors: Dowdy Steven F. , Weissman Bernard E. , Stanbridge Eric J.
Published: February 2005

We report the investigation of the growth properties of tumorigenic and reverted nontumorigenic Wilms' nephroblastoma cells when cultured in serum‐free medium. Wilms' tumor, a pediatric nephroblastoma, has been associated with deletions encompassing the p13 band of chromosome 11 and an independent loss of heterozygosity at 11p15. Weissman et al. (Science 236:175–180, 1987) transferred a human der(11) chromosome into the G401.6TG.6 Wilms' tumor cell line via the microcell‐mediated chromosome transfer technique. The resulting microcell hybrids were nontumorigenic when assayed in nude mice; however these cells retained all of the in vitro growth and morphological characteristics of the tumorigenic parental cells in 10% fetal calf serum (FCS). Segregation of the der(11) Chromosome from the nontumorigenic microcell hybrid cells resulted In the reappearance of the tumorigenic phenotype in vivo. In vitro culture of these cell lines in serum‐free medium supplemented with 0.1% bovine serum albumin (BSA) and 10 ng/ml Na2O3Se resulted in sustained growth of both the tumorigenic parent and the tumorigenic segregant while the nontumorigenic microcell hybrids were unable to divide. The separate addition of either 10 ng/ml of epidermal growth factor (ECF) or 5 μg/ml of insulin did not alter this effect. However, the addition of 5 μg/ml of transferrin stimulated the nontumorigenic microcell hybrid cells to grow at a rate comparable to the tumorigenic cells. In addition, conditioned serum‐free medium from the tumorigenic parental or tumorigenic segregant cell lines was able to stimulate the growth of the nontumorigenic microcell hybrid cells, whereas the reciprocal experiment had no effect on the growth of the tumorigenic cells. These data suggest that the inability of the microcell hybrid cells to grow in serum‐free conditions is correlated with their genetic nontumorigenic phenotype and that a specific growth factor, transferrin, can bypass or alter this negative growth regulatory pathway(s) in vitro.
Effect of recombinant human interleukin‐6 on nitrite production of mouse myeloid leukemia cells
Authors: Takagi Kuniaki , Hosaka Tsutomu , Suketa Yasunobu
Published: February 2005

The effect of recombinant human interleukin‐6 (rhlL‐6) on induction of nitrite (NO) production was investigated in a mouse myeloid leukemia cell line (M1) and a subclone (Mm1). NO was induced by rhlL‐6 (>50 U/ml) in these cell lines. Pretreatment with rhlL‐6 (100 U/ml) for 1 day synergistically enhanced the production of NO in Mm1 by LPS. Furthermore, pretreatment withl lL‐6 (100 U/ml) shortened the lag time of induction. These results indicate that lL‐6 is involved in the regulation of the NO production in macrophage‐like cells.
Association of c‐myc protein with enzymes of DNA replication in high molecular weight fractions from mammalian cells
Authors: Studzinski George P. , Shankavaram Uma T. , Moore Dorothy C. , Reddy Prem Veer
Published: February 2005

Oncoprotein c‐myc is expressed in proliferating but not quiescent mammalian cells, and its overexpression or inappropriate expression is associated with malignant transformation. However, in spite of an intense interest, the normal function of this protein has remained elusive. As a step towards the elucidation of the function of c‐myc protein, we studied its distribution within several types of cells, including HL 60, K 562, COLO 320, and CHEF/18 cells. In all of the cells studied, c‐myc protein was detected in high molecular weight protein fractions, in 350–600 Kd range, in gel‐exclusion chromatography and sucrose gradient centrifugation. This distribution of c‐myc protein coincided with the distribution of DNA polymerase α and several other enzymes necessary for DNA replication. The data suggest that c‐myc product may be a component of the replitase complex of enzymes involved in nuclear DNA replication.
Sertoli cells in culture secrete paracrine factor(s) that inhibit peritubular myoid cell proliferation: Identification of heparinoids as likely candidates
Authors: Tung Pierre S. , Fritz Irving B.
Published: February 2005

Conditioned medium from Sertoli cells, prepared from testes of 20‐day‐old rats, contains component(s) that inhibit the incorporation of [3H]‐thymidine into DNA of peritubular myoid cells (PMC) and inhibit the proliferation of PMC. These components are trypsin‐resistant, heat‐stable compounds having a molecular weight less than 30,000. The active inhibitory components in Sertoli cell conditioned medium are inactivated by treatment with heparinase, but not by treatment with hyaluronidase or chondroitin sulfate lyases. Addition of heparin or heparan sulfate results in inhibition of DNA synthesis by PMC in a dose‐dependent manner, whereas other glycosaminoglycans (GAGs) examined (hyaluronic acid, keratan sulfate, and chondroitin sulfate) have no detectable effects. Heparin and heparan sulfate are unique among GAGs tested in inhibiting the characteristic multilayer growth pattern of PMC following the attainment of confluence in serum‐rich medium. On the basis of these and other data presented, it is concluded that heparin and other heparin‐like GAGs synthesized by Sertoli cells are implicated in the modulation of growth of PMC in vitro during co‐culture. It is postulated that heparin may play a similar role in maintaining the quiescent peritubular myoid cell phenotype in vivo.
Increased glutathione levels in quiescent, serum‐stimulated NRK‐49F cells are associated not with a response to growth factors but with nutrient repletion
Authors: Kang Yu‐Jian , Duane Enger M.
Published: February 2005

Treatment of quiescent cells with serum results concomitantly in an increase in cellular glutathione (GSH) content and growth stimulation. A possible association between the GSH increase and the growth response was examined by studying separately the effects of nutrients and growth factors on the levels of cellular GSH and proliferation of quiescent NRK‐49F cells. The addition of fresh medium with 10% calf serum was found to result in both a twofold increase in cellular GSH and growth stimulation (DNA synthesis and cell proliferation). 10% calf serum alone, without fresh medium, stimulated cell growth but failed to cause a comparable increase in cellular GSH. The addition of fresh medium without 10% serum, and of 0.5 mM cysteine and glutamate, resulted in both instances in a marked increase in cellular GSH, but failed to stimulate cell growth, EGF, in contrast, induced a complete mitogenic response but did not increase cellular GSH. Finally, pretreatment with L‐buthionine‐(S, R)‐sulfoximine (BSO), a specific inhibitor of GSH synthesis, decreased cellular GSH and inhibited EGF‐induced DNA synthesis, but these two responses do not, in their dose dependency, correlate. The results obtained thus show that the increase in cellular GSH that occurs in quiescent, serum‐stimulated NRK‐49F cells is a result of nutrient repletion rather than mitogenic stimulation, and increased GSH levels do not necessarily precede DNA synthesis and mitosis.
Pathway of glutamate oxidation and its regulation in the HuH13 line of human hepatoma cells
Authors: Matsuno Tetsuya
Published: February 2005

Well‐coupled mitochondria were isolated from a HuH13 line of human hepatoma cells and human liver tissue. The liver mitochondria showed a feeble glutamine oxidation activity in contrast to the hepatoma mitochondria, whereas they utilized glutamate well for the oxidative phosphorylation. In the liver mitochondria, glutamate was cxidized via the routes of transamination and deamination. On the other hand, glutamate oxidation was initiated preferentially via transamination pathway in the tumor mitochondria. In the liver mitochondria, bicarbonate nearly at a physiological concentration inhibited oxygen uptake with glutamate as substrate. The interaction of bicarbonate with the pathway of glutamate oxidation occured primarily at the level of succinate dehydrogenase, due to competitive inhibition of the enzyme by the compound. In contrast to the liver mitochondria, glutamate oxidation was not affected by bicarbonate in the tumor mitochondria. These results indicate that the aberrations in the glutamate metabolism and its regulation observed in the hepatoma mitochondria may be favorable to the respiration utilizing glutamine and/or glutamate as an energy source.
Continuous activation of primitive hematopoietic cells in long‐term human marrow cultures containing irradiated tumor cells
Authors: Otsuka Teruhisa , Keith Humphries R. , Hogge Donna E. , Eaves Allen C. , Eaves Connie J.
Published: February 2005

Human hematopoietic cells can be maintained in vitro for many weeks in the absence of exogenously provided hematopoietic growth factors if an adequate stromal cell containing adherent layer is present. We have now extended the use of this type of long‐term culture (LTC) system to create a model of perturbed hematopoiesis in which human tumor cells that constitutively produce a variety of factors are co‐cultured together with normal human marrow cells. In the present study, we used the human bladder carcinoma cell line (5637) because these cells were known to produce not only a variety of factors active directly on hematopoietic cells but also factors that can stimulate hematopoietic growth factor production by human marrow stromal cells. Analysis of mRNA extracted from the adherent layer and measurement of growth factor bioactivity in the medium of established LTC of human marrow containing irradiated 5637 cells, showed increased levels of interleukin‐1 and ‐6, as well as granulocyte and granulocyte‐macrophage colony‐stimulating factor production by comparison to control cultures. As in normal cultures, high proliferative potential clonogenic hematopoietic cells were found almost exclusively in the adherent layer of these co‐cultures, but these primitive cells were maintained in a state of continuous turnover, in contrast to control cultures where the same cell types showed the expected oscillation between a quiescent and a proliferating state following each weekly change of the medium. A similar perturbation of primitive progenitor cycling was achieved by adding medium conditioned by 5637 cells twice a week to otherwise normal LTC. The presence of irradiated 5637 cells in the LTC or the addition of 5637 conditioned medium also resulted in modest (2‐ to 3‐fold) but sustained increases in the total hematopoietic progenitor population, as well as in the final output of terminally differentiated granulocytes and macrophages. These findings indicate that primitive hematopoietic cells in LTC can be kept in a state of continuous activation for many weeks by appropriate endogenous or exogenous hematopoietic growth factor provision and that this does not necessarily lead either to their rapid exhaustion or to a large amplification in output of mature progeny.
Reduction of TGF‐beta activity abrogates growth promoting tumor cell‐cell interactions in vivo
Authors: Theodorescu D. , Caltabiano M. , Greig R. , Rieman D. , Kerbel R. S.
Published: February 2005

We have shown in previous studies that metastatically‐competent variant subpopulations (B5, C1) derived from a non‐metastatic murine mammary adenocarcinoma (SP1) have a pronounced growth advantage over their non‐metastatic tumor cell counterparts in primary tumors. As a result, primary tumors can be progressively overgrown by cells having the competence to spread elsewhere in the body. This occurs despite any evidence to indicate an intrinsic in vivo growth rate advantage of the metastatic cells when grown as isolated populations. This suggested that cell‐cell interactions between metastatic and non‐metastatic tumor populations may be involved in the metastatic cell growth dominance process. Evidence was therefore sought for growth factors released by SP1 cells which could preferentially stimulate the B5 or C1 variants and thereby mediate this cell‐cell interaction process. We found that cocultures of SP1 and C1 or B5 cells with irradiated C1, B5, or SP1 “feeder” cells showed significant stimulation of C1 and B5 by SP1 “feeder” cells. Cell growth stimulation in response to EGF, TGF‐α, TGF‐β1, bFGF, PDGF, NGF, IGF‐1, or IGF‐2 demonstrated that only TGF‐β1 could duplicate this effect. A repeat of the coculture experiment in the presence of specific neutralizing anti‐TGF‐β antibodies was therefore undertaken and this was found to markedly reduce the stimulation of C1 or B5 cells by irradiated SP1 cells. Conditioned media from the SP1 and C1 cell lines was quantitated for TGF‐β activity and contained 4.5 ng/ml and 2.0 ng/ml, respectively. However, the majority of the TGF‐β released by SP1 cells was found to be spontaneously active, whereas 70% of the TGF‐β released by C1 cells was in its latent form. Scatchard analysis revealed approximately four times the number of TGF‐β receptors, of similar type and affinity, present on C1 as compared with SP1 cells. The in vitro results support the hypothesis that active TGF‐β released by SP1 cells may stimulate the proliferation of metastatic variant cells in a paracrine like fashion. In vivo evidence for this was obtained by showing that coinjection of irradiated SP1 cells could selectively stimulate tumor growth of viable C1 cells and this effect was markedly diminished by neurtralizing polyclonal anti‐TGF‐β antibodies. Taken together, the results suggest a novel role for TGF‐β in clonal evolution of malignant tumor growth and as a molecular mediator of tumor cell‐tumor cell interactions involved in facilitating tumor progression.
Regulation of interleukin‐6 expression in the lymphoma cell line OCI‐LY3
Authors: Yee C. S. , Messner H. A. , Minden M. D.
Published: February 2005

Previously we described a cell line OCI‐LY3 derived from a patient with non‐Hodgkin's lymphoma. The cell line produced interleukin‐6 (IL‐6) mRNA and protein and demonstrated an autocrine pattern of growth for IL‐6. Southern blot analysis of the IL‐6 gene did not reveal any rearrangement. To determine whether the production of IL‐6 by OCI‐LY3 was due to subtle changes in the promoter of IL‐6 or due to the expression of trans‐acting factors chloramphenicol acetyltransferase (CAT) reporter constructs containing from ‐1, 180 to + 13 to ‐112 to + 13 of a normal IL‐6 gene were elecroporated into the cell line. When these constructs are transferred into unstimulated fibroblasts, no CAT activity is seen; however, CAT activity is induced when the cells are stimulated with either IL‐1α, lipopolysaccharide (LPS), or cyclic adenosine monophosphate (cAMP) analogues. When the cell line OCI‐LY3 was transfected with these constructs, CAT activity was observed; it was not necessary to stimulate the cells with exogenous factors to observe this activity. No CAT activity was observed in a second lymphoma cell line, OCI‐LY13.1, that does not produce IL‐6. These results suggest that the constitutive production of IL‐6 by the cell line OCI‐LY3 is due to the presence of trans‐acting factors that stimulate the expression of IL‐6 and not due to a cis‐acting mutation of the IL‐6 promoter.
Induction of heat‐shock response and alterations of protein phosphorylation by a novel topoisomerase ii inhibitor, withangulatin A, in 9L rat brain tumor cells
Authors: Lee Wen‐Chuan , Lin Kae‐Yuan , Chen Chiu‐Ming , Chen Zong‐Tsi , Liu Hon‐Ju , Lai Yiu‐Kay
Published: February 2005

Withangulatin A is a newly identified in vitro topoisomerase II inhibitor isolated from the Chinese antitumor herb Physalis angulata. In vivo, it was found to be cytotoxic, capable of suppressing general protein synthesis and of inducing the synthesis of a small set of proteins including those generated by heat‐shock treatment. The 70 kDa protein generated by withangulatin A was unequivocally identified as the heat‐shock protein70 (HSP70) since both proteins migrated to the same position on two‐dimensional polyacrylamide gels, could be recognized by a monoclonal antibody to human HSP70, and exhibited identical peptide maps. The induction of protein synthesis by withangulatin A was regulated at the transcriptional level since it was aborted in cells pre‐treated with actinomycin D. However, the initiation of this process did not require de novo protein synthesis since it was not affected by cycloheximide. Other cellular effect of withangulatin A was alterations of protein phosphorylation including an enhancement of phosphorylation of a 32 kDa protein which was also detected in the heat‐shocked cells. Morevoer, this process was observed within 7.5 min after the initial heat treatment which is much faster than the onset of HSP synthesis. Therefore, increased phosphorylation of the 65 kDa protein may represent on of the earliest signals generated by both heat‐shock and withangulatin A and may be involved in the upstream regulation of heat‐shock response in cells.
Effect of tunicamycin on glycosylation of a 50 kDa protein and thermotolerance development
Authors: Lee Yong J. , Kim Dooha , Hou Zi‐Zheng , Corry Peter M.
Published: February 2005

We investigated whether or not a 50 kDa glycoprotein might play an important role in protein synthesis‐independent thermotolerance development in CHO cells. When cells were heated for 10 min at 45.5°C, they became thermotolerant to a heat treatment at 45.5°C administered 12 hr later. The thermotolerance ratio at 10−3 isosurvival was 4.4. The cellular heat shock response leads to enhanced glycosylation of a 50 kDa protein. The glycosylation of proteins including a 50 kDa glycoprotein was inhibited by treatment with various concentrations of tunicamycin (0.2–2 μg/ml). The development of thermotolerance was not affected by treatment with tunicamycin after the initial heat treatment, although 2 μg/ml tunicamycin inhibited glycosylation by 95%. However, inhibiting protein synthesis with cycloheximide (10 μg/ml) after the initial heat treatment partially inhibited the development of thermotolerance. Nevertheless, there was no further reduction of thermotolerance. development by treatment with a combination of 2 μg/ml tunicamycin and 10 μg/ml cycloheximide. These data suggest that development of thermotolerance, especially protein synthesis‐independent thermotolerance, is not correlated with increased glycosylation of the 50 kDa protein.
Recessive (mediator−) revertants from c‐h‐ras oncogene‐transformed NIH 3T3 cells: Tumorigenicity in nude mice and transient anchorage and serum independence of the recovered tumor cells in culture
Authors: Omata‐Yamada Toshiko , Yamada Hisafumi , Lengyel Peter
Published: February 2005

We have reported earlier the isolation of two recessive, serum‐ and anchoragedependent revertants (R116 and R260) from a c‐H‐ras oncogene‐transformed NIH 3T3 line. In both revertants, the oncogene was fully expressed and fusion of either revertant with (untransformed) NIH 3T3 cells, or of the two revertants with one another, resulted in transformed progeny. These, and other data, indicated that the transforming activity of the oncogene was impaired in the two revertants in consequence of defects in distinct genes needed to mediate this activity.

We report here that neither revertant could be re‐transformed by the K‐ras or N‐ras oncogene (though they could be re‐transformed by several other oncogenes). The two revertants turned out to be tumorigenic in nude mice (though less so than the parental transformed cells). The tumor cells, as recovered, formed foci and had a transformed morphology and a greatly diminished serum and anchorage dependence. Growth of the cells in culture (for 20 passages) resulted in their regaining the characteristics (i.e., anchorage and serum dependence) of cultured R116 and R260 cells. Proliferation of the cells in nude mice was not accompanied by a change in the level of ras oncogene expression or in gene amplification, at least as manifested in the lack of appearance of double‐minute chromosomes. The addition of the growth factors TGF alpha and beta to the medium of either revertant did not support anchorage‐independent growth.
Multiple pathways for ligand internalization in rat hepatocytes I: Effects of anoxia, phenylarsine oxide and monensin
Authors: Moss Anita L. , Ward Walter F.
Published: February 2005

It has been suggested that there are multiple pathways for the cellular internalization of insulin. To investigate these pathways we have examined the effects of three perturbations of endocytosis on the insulin internalization process and have compared these effects with those obtained using an asialoglycoprotein, asialofetuin (Afet), and epidermal growth factor (EGF). Freshly isolated hepatocytes were incubated with radiolabeled ligands and internalization measured under conditions of anoxia to deplete cellular ATP, in the presence of phenylarsine oxide (PAO) to inhibit endocytosis, and in the presence of monensin to interfere with endosomal acidification. Afet internalization essentially was blocked by all three treatment processes, while insulin internalization was inhibited approximately 40% in the presence of anoxia, and 54% in the presence of PAO. Monensin exhibited differential effects on internalization of high and low insulin concentrations. The effects of the treatment processes on EGF internalization were intermediate to those seen with Afet and insulin. These results suggest that insulin and EGF utilize routes of internalization exhibiting different energy requirements that may correspond to coated pit, non‐coated pit, and fluid‐phase internalization pathways. The observations with Afet internalization remain consistent with utilization of the coated pit pathway.
Inhibition of protein synthesis and heat protection: Histidinol‐resistant mutant cell lines
Authors: Lee Yong J. , Hou Zi‐Zheng , Kim Dooha , Al‐Saadi Abdul , Corry Peter M.
Published: February 2005

The mechanism of histidinol (HST) ‐induced heat protection was investigated to test the hypothesis that the cessation of protein synthesis itself is one of the events involved in heat protection. For this study, we isolated three HST‐resistant mutant strains. HST (5 mM), which inhibited protein synthesis by 88% in the wild type, caused only 0, 9, and 25% inhibition in three mutants, respectively. The drug, which afforded heat protection, (i.e., a 125‐fold increase in survival from 4 x 10−3 to5 x 10−1 after 2 hr at 43°C in wild type), did not protect mutant cells from heat killing. In contrast, cycloheximide (10 μg/ml) which inhibited protein synthesis by 95% in both wild type and mutant cell types, protected both cell types from heat killing. Therefore, these results suggest that the cessation of protein synthesis, per se, preventing synthesis of nascent polypeptides, is a major event leading to heat protection.
An IGF binding protein is an inhibitor of fgf stimulation
Authors: Villaudy J. , Delbé J. , Blat C. , Desauty G. , Golde A. , Harel L.
Published: February 2005

We purified to homogeneity a growth inhibiting diffusible factor (IDF45) secreted by dense cultures of mouse 3T3 cells and which was able to inhibit 100% of DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF) (Blat et al., 1989a). We then demonstrated that this factor was an IGF‐binding protein (Blat et al., 1989b). Indeed, its N‐terminal amino acid sequence was homologous to that of rat IGFBP‐3. Our present results show that basic fibroblast growth factor (bFGF) induced, respectively, a fivefold and threefold increase in DNA synthesis in mouse embryo fibroblasts (MEF) and CEF. IDF45 inhibited the stimulation induced by bFGF by about 65%, while stimulation induced by insulin, PDGF, or EGF was only weakly or not at all inhibited by IDF45. When bFGF stimulation was determined in the presence of a high concentration of insulin in conditions which minimize the effect of endogenous IGF‐I or ‐II, this stimulation was decreased by about 50% in the presence of IDF45. This result suggests that addition of bFGF stimulates IGF secretion, thereby resulting in partial loss of inhibition, by IDF45, of bFGF stimulation.
Na+/K+/Cl− cotransport is stimulated by a Ca++‐calmodulin–;mediated pathway in BALB/c 3T3 fibroblasts
Authors: Snyder David , Atlan Henri , Markus Miriam , Panet Rivka
Published: February 2005

In the present study, we investigated the role of intracellular Ca++ in the stimulation of the Na+/K+/Cl– cotransport in synchronized BALB/c 3T3 cells. The Na+/K+/Cl– cotransport was stimulated by the growth factors EGF, TGF‐α, IGF‐1, and IGF‐2, which do not activate protein kinase C, but do induce a transient increase in free cytoplasmic Ca++ In addition, direct activation of protein kinase C by the phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) did not affect the Na+/K+/Cl– cotransport activity of quiescent cells. The Na+/K+/Cl– cotransport was also stimulated by the above mitogens in cells pretreated with the phorbol ester TPA. This treatment led to a progressive decline in the activity of cellular protein kinase C. This result implies that cells deficient in protein kinase C may still support stimulation of the Na+/K+/Cl– cotransport. Taken as a whole, these findings suggest that the Na+/K+/Cl– cotransport is stimulated predominantly by a protein kinase C‐independent mechanism in BALB/c 3T3 fibroblasts.

Both the intracellular Ca++ antagonist 8‐(N,N‐diethylamino)octyl‐3,4,5‐trimethoxybenzoate (TMB‐8) and two potent calmodulin antagonists, trifluoperazine (TFP) and chloropromazine(CP), blocked serum‐ and mitogen‐stimulated Na+/K+/Cl– cotransport. These results suggest that the Na+/K+/Cl– cotransport is stimulated by an increase of intracellular Ca++ and subsequently by a Ca++‐calmodulin‐mediated pathway in the synchronized BALB/c 3T3 fibroblasts.
Biologically active synthetic fragments of human basic fibroblast growth factor (bFGF): Identification of two Asp‐Gly‐Arg‐Containing domains involved in the mitogenic activity of bFGF in endothelial cells
Authors: Presta Marco , Rusnati Marco , Urbinati Chiara , Sommer Andreas , Ragnotti Giovanni
Published: February 2005

Synthetic peptides derived from the amino acid sequence of human basic fibroblast growth factor (bFGF) have been assayed for the capacity to exert bFGF agonist and antagonist activities in cultured endothelial cells. bFGF fragments A and C, which correspond to the sequences bFGF (38–61) and bFGF (82–101), induce a limited but statistically significant increase in cell number when administered to cultures of fetal bovine aortic endothelial GM 7373 cells and adult bovine aortic endothelial cells. The two peptides also exert a partial antagonist activity when GM 7373 cells are stimulated to proliferate by bfGF, but they do not affect cell proliferation induced by serum, epidermal growth factor (EGF), phorbol ester (TPA), or 1,2‐diacylglycerol (diC8). Moreover, antibodies raised against peptides A and C specifically quench the mitogenic activity of bFGF. Peptides A and C contain the amino acid sequence Asp‐Gly‐Arg (DGR), which is the inverse of the cell adhesion signal sequence RGD recognized by integrins. DGR‐ and RGD‐containing tetra‐ and heptapeptides inhibit the mito‐genic activity exerted by bFGF and by the two active bFGF fragments. They do not affect cell proliferation induced by acidic FGF, EGF, serum, TPA, and diC8. However, neither peptides A and C, their corresponding antibodies, nor DGR‐and RGD‐containing peptides inhibit the binding of 125I‐bFGF to its low and high affinity binding sites. The data suggest that amino acid residues 38–61 and 82–101, both containing a core DGR sequence, represent two “activation” domains of bFGF. Both domains are involved in the modulation of the mitogenic activity of bFGF without interacting directly with the bFGF receptor.
Loss of sperm in juvenile spermatogonial depletion (jsd) mutant mice is ascribed to a defect of intratubular environment to support germ cell differentiation
Authors: Mizunuma Masumi , Dohmae Kayoko , Tajima Youichi , Koshimizu Uichi , Watanabe Daisuke , Nishimune Yoshitake
Published: February 2005

C57BL/6(B6)‐jsd/jsd mice are sterile due to the defective spermatogenesis in the testes. To know the cause of the deficient spermatogenesis in B6‐jsd/jsd mice, we examined whether the problem is within or outside the seminiferous tubules by transplanting tubules from cryptorchid testes of B6−+/+ mice into B6‐jsd/jsd testes or tubules from B6‐jsd/jsd mice into testes of (WB × C57BL/6)F1‐W/W (hereafter, WBB6F1‐W/W) mice. Type A spermatogonia differentiated into spermatids in seminiferous tubules from cryptorchid testes transplanted into B6‐jsd/jsd testes. In contrast, in B6‐jsd/jsd tubules transplanted into WBB6F1‐W/W testes, type A spermatogonia were stimulated to mitotic proliferation, but didn't proceed to any differentiated germ cells. The present results suggest that the cause of the deficient spermatogenesis in B6‐jsd/jsd mice is a defect of intratubular environment to support germ cell differentiation.
Regulation of NF‐κB activity in murine macrophages: Effect of bacterial lipopolysaccharide and phorbol ester
Authors: Vincenti Matthew P. , Burrell Terrie A. , Taffet Steven M.
Published: February 2005

Nuclear factor kappa‐B (NF‐κB) has been shown to play an important role in LPS‐mediated induction of several genes in macrophages. Several studies have implicated protein kinase C (PKC) or cAMP‐dependent protein kinase in the regulation of NF‐κB activity. In this study we have investigated the mechanism of NF‐κB induction in murine macrophages. A chloramphenicol acetyl transferase (CAT) expression vector containing multiple copies of the TNF‐α NF‐κB element was transfected into the RAW264 macrophage‐like cell line and assessed for inducible CAT activity. LPS treatment of the transfected cells resulted in a significant induction of CAT activity. CAT activity was not induced by treatment with phorbol myristate acetate (PMA) or the cAMP analogue 8‐bromo cAMP. To further study NF‐κB induction, nuclear extracts were prepared from RAW264 cells. Extracts from RAW264 cells that were treated from 30 min to 2 hr with LPS had a significant increase in NF‐κB binding activity as determined by the electrophoresis mobility shift assay (EMSA). Treatment of these cells from 30 min to 2 hr with PMA did not result in such binding activity. U.V. crosslinking analysis of the DNA‐binding activity confirmed these results and indicated that LPS induced a 55 KD DNA‐binding protein. Induction of this NF‐κB binding activity was not inhibited by pretreatment with the PKC inhibitor H‐7. H‐7 did inhibit induction of TPA responsive element binding by either LPS or PMA. Prolonged exposure to phorbol ester, a treatment which down‐regulates PKC, had no effect on LPS induction of NF‐κB activity in these cells. These results suggest that the induction of NF‐κB in macrophages by LPS is independent of PKC.
Identification of basic fibroblast growth factor sensitivity and receptor and ligand expression in human colon tumor cell lines
Authors: New Bonnie A. , Yeoman Lynn C.
Published: February 2005

Basic fibroblast growth factor (bFGF) has been shown to be mitogenic to many different eukaryotic cell lines of mesodermal and neuroectoderrnal origin. Addition of exogenous bFCF to the chemically defined media of five characterized human colon tumor cell lines, cultured in the absence of epidermal growth factor (EGF), resulted in stimulation of growth from 24% to 146% in four of five cell lines, as measured by a colorimetric MTT assay. A positive dose‐response relationship was observed when colon cells were treated with bFGF concentrations from 1 pM to 1 nM. bFGF showed a cumulative effect with EGF in stimulating the proliferation of colon tumor cells. The growth‐inhibitory effect of exogenous transforming growth factor‐beta (TGF‐β) on these cells was abolished by bFGF. When colon tumor cells were examined on immunoblots with a fibroblast growth factor (FGF) receptor‐specific antibody, bands were detected at apparent molecular weights of 131 and 145 kDa. Conditioned media and cell lysates from the same human colon tumor cell lines were immunoprecipitated with a bFGF‐specific antibody. An immunoreactive band was detected that comigrated with authentic human recombinant bFGF (16 kDa). Furthermore, preabsorption of anti‐bFGF antibody with authentic ligand blocked immunodetection of the 16 kDa band on immunoblots. Documentation of a bFGF response, receptor, and ligand expression in human colon tumor cell lines is novel, and may represent a more widespread role for FGF that extends to epithelial cells and tumors of endodermal germ layer origin. The expression of both ligand and receptors by these cells indicates that bFGF could be involved in their growth regulation at the autocrine level.
Increased peptidylarginine deiminase expression during induction of prolactin biosynthesis in a growth‐hormone‐producing rat pituitary cell line, MtT/S
Authors: Nagata Saburo , Uehara Takemi , Inoue Kinji , Senshu Tatsuo
Published: February 2005

Insulin and type I insulin‐like growth factor (IGF‐I) suppressed growth hormone (GH) expression followed by the induction of prolactin (PRL) biosynthesis in MtT/S cells cultured with normal sera. Insulin also increased the peptidylarginine deiminase activity in a dose‐dependent manner. The increase was detectable at 1 ng/ml and reached a maximum (about 16‐fold higher than the control) at 1 μg/ml. IGF‐I showed similar but less prominent effects. The enzyme activity started to increase by 15 hr after the addition of insulin (500 ng/ml), and reached a plateau level at 48 hr. There were concurrent increases in the enzyme mRNA level, enzyme biosynthesis, and enzyme protein contents detected by Northern blot hybridization, [35S]‐amino‐acid incorporation, and Western immunoblot analysis, respectively. Two‐color immunofluorescence staining at 1 day after the insulin addition detected a small number of peptidylarginine‐deiminase‐positive cells (about 1% of the total cells) which were also GH‐positive. The enzyme‐positive cells increased to 12% on day 2 and to 24–26% on days 4–6. PRL‐positive cells first appeared in the enzyme‐positive cell population on day 2, and PRL‐positive, enzyme‐negative cells appeared later. These results suggest that peptidylarginine deiminase expression increases in association with the hormone switching in MtT/S cells. When the cells were cultured in a steroid‐depleted medium, insulin failed to increase the enzyme activity. The insulin action could be specifically restored by estrogen, indicating estrogen‐insulin synergism in regulation of the enzyme expression.
Mapping the entry of reactive oxygen metabolites into target erythrocytes during neutrophil‐mediated antibody‐dependent cellular cytotoxicity
Authors: Petty Howard R. , Liang Bing , Maher Robert J.
Published: February 2005

Transmitted Soret band optical microscopy has been used to image the entry and passage of reactive oxygen metabolites across target erythrocytes. Due to the rapid cytosolic diffusion of hemoglobin in comparison to video rates, it was necessary to use erythrocytes with relatively immobilized hemoglobin. To achieve this, erythrocytes from patients with sickle cell anemia were used. The movement of reactive oxygen metabolites across rabbit IgG‐opsonized sickle cells was observed in real time. These observations indicate that reactive oxygen metabolites can enter and cross targets in an asymmetric fashion.
Thrombin signal transduction mechanisms in human glomerular epithelial cells
Authors: He Ci‐Jiang , Peraldi M. N. , Adida C. , Rebibou J. M. , Meulders Q. , Sraer J. D. , Rondeau E.
Published: February 2005

We have previously shown that α‐thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase‐type (u‐PA) and tissue‐type plasminogen activator (t‐PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI‐1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose‐dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L‐type voltage‐sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin‐stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10−8 M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin‐stimulated synthesis of u‐PA, t‐PA, and PAI‐1. Both thrombin‐stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 μM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP‐inactivated thrombin which binds the thrombin receptor and γ‐thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u‐PA, t‐PA, and PAI‐1 synthesis although to a lesser extent than α‐thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by α‐thrombin in glomerular epithelial cells: (1) Ca2+ influx through a dihydroperidine‐sensitive calcium channel, which seems critical for mitogenesis; (2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u‐PA, t‐PA, and PAI‐1; (3) protein kinase C activation by other phospholipid breakdown can stimulate u‐PA, t‐PA, and PAI‐1 synthesis but not mitogenesis.
Common senescent cell‐specific antibody epitopes on fibronectin in species and cells of varied origin
Authors: Porter Mary Beth , Pereira‐Smith Olivia M. , Smith James R.
Published: February 2005

The phenomenon of in vitro cellular senescence has been demonstrated in cultured cells derived from humans and various other species. We have previously shown that monoclonal antibodies SEN‐1, SEN‐2, and SEN‐3 react to epitopes on fibronectin that are exposed when human diploid fibroblasts become senescent. We here present results demonstrating that exposure of these epitopes is specific to senescence for a variety of human cells: epidermal keratinocytes, mammary epithelial cells, as well as fibroblasts. Fibronectin from 11 additional species was also analyzed by Western immunoblot for ability to bind the SEN antibodies. SEN‐1 bound only human and gorilla fibronectin, whereas SEN‐2 and SEN‐3 bound fibronectin from those two species as well as the horse, cow, sheep, goat, dog, and chick. None of the antibodies reacted with fibronectin from the rabbit, rat, or mouse. These data indicated a correlation between the ability of the SEN antibodies to bind fibronectin from a particular species and the ability of cells from that species to exhibit a stable senescent phenotype in vitro. Therefore, exposure of this region of fibronectin may be important in the establishment and maintenance of cellular senescence. In addition, the ability of the SEN antibodies to react with fibronectin from a variety of senescent cells emphasizes their usefulness as markers for cellular senescence.
Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum‐Golgi complex
Authors: Mignatti Paolo , Morimoto Takashi , Rifkin Daniel B.
Published: February 2005

Basic fibroblast growth factor (bFGF) modulates functions of a variety of cell types. Whereas bFGF is known to act extracellularly, the protein lacks a transient signal peptide. No defined mechanism for bFGF secretion has been characterized besides release from dead or injured cells. To study this problem we devised an experimental system to examine bFGF‐mediated migration of isolated single cells. Under these conditions individual cells are not affected by bFGF derived from other cells. By this method we have previously shown that bFGF released by NIH 3T3 cells transfected with bFGF cDNA modulates migration in an autocrine manner. We have now examined the effects on cell motility of drugs or treatments known to affect various pathways of protein secretion. Drugs that block secretion via the endoplasmic reticulum (ER)‐Golgi complex or via multidrug resistance proteins did not inhibit cell motility. Migration was enhanced by the calcium ionophore A23187, which stimulates exocytosis, and was inhibited by methylamine, serum‐free, and low temperature (18°C) conditions, which block endoand exocytosis. The reversal of these effects by the concomitant addition of affinity‐purified anti‐bFGF IgG or recombinant bFGF showed that the alterations in cell migration were mediated by changes in bFGF externalization. Thus bFGF can be released via a mechanism of exocytosis independent of the ER‐Golgi pathway. © 1992 Wiley‐Liss, Inc.
Differential extracellular matrix gene expression by fibroblasts during their proliferative life span in vitro and at senescence
Authors: Choi Augustine M. K. , Olsen David R. , Cook Keith G. , Deamond Scott F. , Uitto Jouni , Bruce Sarah A.
Published: February 2005

Increasing evidence supports the idea that the finite proliferative life span of normal fibroblasts is a differentiation‐like phenomenon. If this were correct, an ordered sequence of differential gene expression should be associated with the in vitro progression of cells from low passage to high passage (senescence). To define the pattern of expression of fibroblast differentiation‐associated genes during this in vitro progression, we have determined the temporal pattern of expression of extracellular matrix (ECM) genes in Syrian hamster dermal fibroblasts as a function of passage level and percentage of proliferative life span in vitro. Steady‐state mRNA levels were determined by Northern and dot blot analyses of total cellular RNA hybridized with cDNA probes specific for fibronectin, procollagen α1 III, and procollagen α1 I. Cells were analyzed at 24 hr postconfluence to minimize the presence of actively proliferating cells, and because maximal levels of fibronectin, α1 III, and α1 I mRNAs were observed 24 hr postconfluence. Unique, multiphasic patterns of expression of each of these ECM components were observed as the cells progressed from low passage to high passage. As the cells reached midhigh passage, fibronectin mRNA levels increased. This midpassage increase in fibronectin was followed by an increase in the level of α1 III mRNA as the cells reached the end of their in vitro proliferative life span, and then α1 I when the cells entered the postmitotic senescert phase, at which time the level of fibronectin mRNA also declined. A similar overlapping cascade pattern of up‐regulation of these genes is seen during development and wound repair. This suggests that as cultured fibroblasts reach the end of their proliferative life span, they reinitiate a gene expression program used in tissue development and repair. © 1992 Wiley‐Liss, Inc.