摘要:

AbstractThe fibroblast growth factor receptor 1 (flg) contains eight acidic amino acids between the first and second immunoglobulin domain. This report examines the role of the acidic domain in the interaction of the flg receptor with its ligands. We observed a marked inhibition of binding of bFGF to the receptor when the acidic domain was completely deleted, but mutants with two and four amino acids deleted (flgΔ2and flgΔ4, respectively) still bound the ligand. After addition of a bifunctional cross‐linking reagent, cross‐linked complexes (between bFGF and receptor) with the expected size were observed in cells expressing mutants lacking two or four acidic residues, but not in cells expressing mutants lacking six or eight acidic residues. Immunoprecipitation with anti‐flg antibody followed by electrophoresis produced a band of 90 Kd in tunicamycin‐treated cells expressing the mutant as well as the wild‐type receptors, indicating that the inhibition of binding was not due to defective expression of the protein. The ability of flgΔ8to mediate a mitogenic response to FGFs was also greatly reduced when this mutated receptor was expressed in receptor‐negative cells. The effect of replacing the acidic amino acids with lysine residues was also studied. Binding of bFGF to cells transfected with a plasmid encoding a mutated protein with four amino acid substitutions was totally inhibited, but an eight amino acid substitution did not alter ligand binding to the receptor. In this case the mutation with four amino acids substitution caused a drastic impairment of protein expression. Thus the acidic domain of the FGFR‐1 plays an essential role in receptor function, either because it is important for a stable protein configuration or for ligand‐receptor interaction. © 1

ISSN:0021-9541    

DOI:10.1002/jcp.1041570202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractTo better understand the mechanism by which insulin exerts effects on events at the cell nucleus, we have studied insulin receptors and tyrosine kinase activity in nuclei isolated by sucrose density gradient centrifugation following insulin treatment of differentiated 3T3‐F442A cells. Insulin stimulated nuclear accumulation of insulin receptors by approximately threefold at 5 min. The half‐maximal effect was observed with 1–10 nM insulin. Following insulin treatment, phosphotyrosine content associated with the nuclear insulin receptor was also increased by twofold at 5 min with a similar insulin concentration dependency. These nuclear insulin receptors differ from the membrane‐associated insulin receptors in that they were not efficiently solubilized with 1% Triton X–100. During the same period of time, insulin stimulaced nuclear tyrosine kinase activity toward the exogenous substrate poly Glu4: Tyr1tenfold in a time‐dependent manner reaching a maximum at 30 min. The insulin receptor substrate protein 1 (IRS‐1) could not be detected in the nucleus by immunoblotting. However, a nuclear protein with Mr≈ 220 kDa was tyrosine phosphorylated, and insulin further stimulated this process threefold>30 mins. Surface labeling was performed to determine if the nuclear insulin receptors would emerge from the plasma membrane fraction. Using1251‐BPA‐insulin with intact cells, the intensity of nuclear insulin receptor labeling was negligible and not increased throughout 30 min incubation at 37°C. In contrast, there was an increase in labeled receptors in the microsomal fraction following insulin treatment. Taken together, these results indicate that insulin rapidly increases nuclear insulin receptor appearance and activates nuclear tyrosine kinase activity. The insulin‐induced accumulation of nuclear insulin receptors cannot be accounted for by internalization of surface membrane receptors. These effects of insulin may play an important role in action of the hormone at the nuclear level.

ISSN:0021-9541    

DOI:10.1002/jcp.1041570203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe insulin‐like growth factors (IGFs) are potent mitogens for breast cancer cells and their activity is modulated by high affinity binding proteins (IGFBPs). We have recently shown that IGFBP‐1 purified from human amniotic fluid neutralizes IGF‐I‐dependent growth of MCF‐7 cells. In this study we examined the effects of recombinant IGFBP‐1 (rBP‐1) on IGF‐I, estradiol (E2), and serum‐induced monolayer and anchorage independent growth (AIG) of MCF‐7 cells. Under serum‐free conditions, rBP‐1 had no effect on MCF‐7 basal monolayer growth. However, 40 nM rBP‐1 completely blocked the mitogenic action of both IGF‐I and 5% charcoal stripped serum (CSS). This concentration of rBP‐1 partially inhibited E2‐induced growth, while 80 nM rBP‐1 completely abolished E2mitogenicity. The addition of either excess IGF‐I or 5 nM [Arg3]IGF‐I, a species that does not bind IGFBPs, neutralized rBP‐1 inhibitory effects. In AIG assays, 80 nM rBP‐1 reduced colony number by at least 70% and decreased colony size in all treatment groups compared to control. We examined rBP‐1 effects on both IGF‐I binding to MCF‐7 membranes and activation of type I IGF receptor (IGFR1) and found that 80 nM rBP‐1 reduced IGF‐I receptor binding to levels of nonspecific binding and completely abolished ligand‐dependent IGFR, phosphorylation. However, neither treatment with 5% CSS nor exposure to E2resulted in IGFR1phosphorylation suggesting that different mechanism(s) are responsible for rBP‐1 inhibitory action under this condition. Our data suggest rBP‐1 may serve as an antagonist of human breast cancer growth by interfering with growth

ISSN:0021-9541    

DOI:10.1002/jcp.1041570204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe epidermal growth factor (EGF) anderbB‐2 receptors are structurally related membrane‐bound tyrosine kinases. While these proteins exhibit close sequence homology, 50% overall and 80% in the tyrosine kinase domains, they respond very differently to heat stress. In NIH‐3T3 or NR6 cells transfected with wild‐type EGF‐R and incubated at 37°C or heat shocked at 46°C, EGF binds to its receptor and stimulates receptor autophosphorylation to equivalent extents. At 46°C, however, the basal tyrosine kinase activity of the wild‐typeerbB‐2 receptor is rapidly lost. When cells containing chimeric receptors composed of the EGF‐R extracellular domain and intracellular domain oferbB‐2 were heat stressed,125I‐EGF bound to the receptors, but did not stimulate receptor autophosphorylation. The decline in EGF‐stimulated chimericerbB‐2 receptor autophosphorylation is dependent on the length of heat shock, with nearly 100% of the kinase activity lost after 60 min at 46°C. The loss of chimeric receptorerbB‐2 kinase activity is not due to degradation of receptor protein, nor is it attributable to a specific transmembrane domain from either the EGF orerbB‐2 receptors. Sensitivity oferbB‐2 to heat stress is also not a result of denaturation of this receptor's carboxy‐terminal domain. Insertion of theerbB‐2 tyrosine kinase domain into the EGF‐R confers heat stress sensitivity to the resultant chimeric receptor. Thus, although the EGF‐R anderbB‐2 kinase domains show a high degree of homology, the secondary/tertiary structures of these domains would seem to be stabilized

ISSN:0021-9541    

DOI:10.1002/jcp.1041570205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe aim of this study was to determine the role of ECM components of bone in regulating the differentiation and function of cells of the osteoblast lineage. Rat UMR 201 cells, phenotypically preosteoblast, were plated onto plastic tissue culture dishes or dishes coated with gelled type I collagen or reconstituted basement membrane (matrigel). Acute cell attachment assays showed that cells adhered to substrates in the following order: collagen>matrigel ≫ plastic. Proliferation rate up to 96 hr were similar on each substrate. However, if cells were treated with 10−6M retinoic acid (RA), proliferation rates were reduced compared with control for cells grown on collagen and matrigel but not on plastic. Morphological changes were matrix‐specific; in subconfluent cultures, long thin processes were seen with cells grown on collagen and a pattern of interconnecting cell processes formed when cells were plated on matrigel. Striking differences were observed in the constitutive or RA‐induced gene expression of cells grown on the different substrates. When cells plated on collagen were treated with RA, induction of mRNA for alkaline phosphatase (ALP) as well as ALP enzyme activity were much less than with cells grown on plastic. In contrast, RA treatment induced osteopontin (OP) mRNA expression more strongly in cells plated on collagen compared with plastic within 24 hr and this was maintained for 72 hr. RA treatment produced a two fold increase of pro‐α 1(I) collagen mRNA in cells grown on plastic and matrigel but not in cells grown on collagen. Growth on collagen produced changes in the way UMR 201 cells responded to RA from which they did not fully recover in subsequent 48‐hr growth periods on plastic. These results indicate that ECM components regulate the function of and are capable of modulating RA‐induced differentiation of preosteoblasts. © 1993

ISSN:0021-9541    

DOI:10.1002/jcp.1041570206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

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

ISSN:0021-9541    

DOI:10.1002/jcp.1041570207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

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

ISSN:0021-9541    

DOI:10.1002/jcp.1041570208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractVitamin D3metabolites regulate the differentiation of chondrocytes isolated from the growth zone or resting zone of rat costochondral cartilage. Since some of the direct membrane effects of vitamin D metabolites are nongenomic, we hypothesized that protein kinase C (PKC) plays a role in signal transduction for these chondrocyte differentiation factors and that the regulation of PKC by the vitamin D metabolites is cell maturation dependent. Confluent, fourth passage cultures of growth zone and resting zone chondrocytes were treated with vitamin D3metabolites for up to 24 h, lysed, and cell extracts assayed for kinase activity using a specific PKC substrate peptide. The addition of 1,25‐(OH)2D3to growth zone cell cultures resulted in a rapid dose‐dependent stimulation of PKC, significant at 10−9‐10−7M, beginning at 3 min and sustained until 90 min; 1,25‐(OH)2D3had no effect on PKC activity in resting zone chondrocyte cultures. The addition of 24,25‐(OH)2D3to resting zone cultures showed a slower PKC activation, with significant stimulation seen at 90–360 min for 10−8‐10−7M 24,25‐(OH)2D3. However, 24,25‐(OH)2D3had no effect on PKC activity in growth zone cell cultures at all times and concentrations examined. The specificity of PKC stimulation by the vitamin D3metabolites was verified using a specific pseudosubstrate region peptide inhibitor, which reduced PKC activity when included in the reaction mixture. Pretreatment of the cultures with U73,122, a phospholipase C inhibitor, decreased 1,25‐(OH)2D3—stimulated PKC activity but had no effect upon 24,25‐(OH)2D3–induced activity. The tyrosine kinase inhibitor, genistein, did not inhibit the PKC response in either vitamin D3metabolites‐treated culture. Neither actinomycin D nor cycloheximide affected 1,25‐(OH)2D3–induced PKC activity in growth zone chondrocyte cultures, while both compounds inhibited 24,25‐(OH)2D3—indiuced activity in resting zone chondrocyte cultures. The results of this study indicate that vitamin D metabolites stimulate PKC activity in a metabolite‐ and cell‐maturation‐specific manner. Effects of 1,25‐(OH)2D3appear to be nongenomic, whereas the effects of 24,25‐(OH)2D3probabl

ISSN:0021-9541    

DOI:10.1002/jcp.1041570209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractBovine aortic endothelial cells (BAEC) can be isolated in large numbers without major contamination by other cells and maintained in culture with a limited life span for about 100 population doublings. In order to study phenotypic changes of BAEC during long‐term culture, stocks of different passages of BAEC were established and their morphological, migratory, and proliferative properties analyzed. Early‐passage BAEC (passages 5–15) rapidly produce dense, cobblestone‐like monolayers. Their growth beyond the monolayer configuration is characterized by the formation of an irregular network of spindle‐shaped, crisscrossing BAEC growing either on top or beneath the monolayer, and by the assembly of elongated BAEC into well‐differentiated capillary‐like tubes. In contrast, senescent BAEC (passages 35–45) form perfect cobblestone monolayers that contain several, often multinucleated giant cells and a few capillary‐like tubes but not the crisscrossing networks of their early‐passage counterparts. The rates of BAEC migration and proliferation gradually decline during in vitro senescence. This decline is neutralized by exogenous basic fibroblast growth factor (bFGF) which elevates the migratory and proliferative capacities of early‐passage and senescent BAEC to uniformly high levels. Northern blot analysis shows a gradual decline in bFGF message and an increase in laminin message during in vitro BAEC senescence. The present study supports the concept of autocrine growth regulation of BAEC and associates a decreased bFGF message with decreased rates of migration and proliferation as well as loss of the crisscrossing BAEC morphotype in senescent cultures.

ISSN:0021-9541    

DOI:10.1002/jcp.1041570210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractProhibitin, a novel intracellular antiproliferative protein, blocks entry into the S phase of the cell division cycle when its mRNA is microinjected into normal fibroblasts or HeLa cells. To learn more about the interaction between prohibitin and the cell cycle, we studied the effect of microinjecting prohibitin mRNA at different points during the transition from G0to S phase and analyzed prohibitin mRNA and protein levels in different parts of the cell cycle. The antiproliferative activity of microinjected prohibitin mRNA is high in G0/G1and falls as cells approach S phase. Prohibitin mRNA and protein levels are high in G1, fall with S phase, rise again in G2, and fall in M. Together, these findings suggest that endogenous prohibitin contributes to the control of the G1to S transition in cycling cells in a complex manner, which involves both a transcriptional and posttranslational mechanism. © 1993 Wiley‐Liss, I

ISSN:0021-9541    

DOI:10.1002/jcp.1041570211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY