摘要:

AbstractF9 teratocarcinoma stem cells differentiate into parietal endoderm‐like cells when given retinoic acid (RA) and dibutyryl cyclic adenosine monophosphate (DB‐cAMP). It is generally accepted that the stem cells are resistant to the action of cAMP alone and need to be primed by RA in order to respond to cAMP. In this report, we demonstrate that F9 stem cells differentiate into parietal endoderm‐like cells in the absence of exogenous RA when treated with cholera toxin and 1‐methyl,3‐isobutyl xanthine (CT/MIX) or 8‐bromo‐cAMP/MIX (8B2‐cAMP/MIX). Cells treated with CT/MIX or 8B2‐cAMP/MIX were morphologically similar to parietal endoderm‐like cells, produced high amounts of plasminogen activator, and synthesized both type IV collagen and laminin mRNA. Conversely, markers made in abundance by stem cells such as stage‐specific embryonic antigen (SSEA‐1) and an mRNA species of 6.8 kb (pST6‐135) were markedly reduced in CT/MIX‐treated cells. To prove that cAMP alone could induce differentiation Lipidex‐1000, a hydrophobio gel, was used to remove 80–;90% of the endogenous serum retinoids. F9 cells grown in this retinoid‐depleted serum and treated with 8B2‐cAMP/MIX differentiated to parietal endoderm‐like cells as shown by both dramatic changes in morphology and induction of type IV collagen mRNA. Our results indicate that the differentiation of F9 to parietal endoderm‐like cells can be induced by increased intracellular cAMP and is not s

ISSN:0021-9541    

DOI:10.1002/jcp.1041430202

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

年代:1990

数据来源: WILEY

摘要:

AbstractRat calvaria osteoblasts derived from 21‐day‐old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al:Biochem. Biophis. Res. Commun.148:1129, 1987) and is similar to that observed in cultures of subcultivated 16‐day‐old embryonic chick calvaria‐derived osteoblasts (COB) (Gerstenfeld, et. al:Dev. Biol.122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of β‐glycerol‐phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200‐fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/mat

ISSN:0021-9541    

DOI:10.1002/jcp.1041430203

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

年代:1990

数据来源: WILEY

摘要:

AbstractNormal human cells such as human diploid fibroblasts (HDF) have a finite pro‐liferative lifespan in culture. Previous studies have shown that the limited lifespan phenotype is dominant in cell hybrids formed by fusion of HDF to at least 23 different kinds of immortal human cells. However, two independent studies reported that hybrid clones formed by the fusion of HDF to the HeLa variant D98 had unlimited division potential. Those results were potentially very important because they implied that a) there is a dominant mechanism for immortalization of human cells in addition to the well‐documented recessive mechanism, and b) a dominant mechanism would lend itself to identification of the immortalizing gene. Consequently, we carried out more detailed studies of the behavior of D98 cells in hybrids. Our results indicate that the majority of D98 x HDF hybrid clones exhibit a clear‐cut finite proliferative lifespan phenotype. In addition, these hybrid cell populations often give rise to an immortal focus of cells that can be seen to take over the population of mortal cells at the end of their lifespan. This phenomenon reconciles our data with the previous reports of immortal D98 x HDF hybrid clones and leads us to conclude that D98 cells do not express a dominant immortalizing

ISSN:0021-9541    

DOI:10.1002/jcp.1041430204

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe DF3 gene codes for a high molecular weight human breast tumor‐associated glycoprotein. The detection of this antigen in human milk has also suggested that its expression represents a differentiated function of mammary epithelium. The present studies have examined the regulation of DF3 gene expression in human MCF‐7 breast carcinoma cells. These cells express two DF3 transcripts of 4.5 and 7.0 kb. Treatment of MCF‐7 cells with 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) was associated with increases in levels of both DF3 mRNAs. When nuclear run‐on assays were used, DF3 gene transcription was at low to undetectable levels in untreated MCF‐7 cells and was increased after TPA exposure. TPA‐induced increases in DF3 mRNA levels were also inhibited by actinomycin D (ACT). MCF‐7 cells exposed to ACT further demonstrated that the half‐lives of the 4.5 and 7.0 kb transcripts are 26 and 11 h, respectively. The results also demonstrate that the protein synthesis inhibitor, cycloheximide (CHX), increases DF3 mRNA levels in MCF‐7 cells. These effects of CHX were sensitive to actinomycin D and not associated with stabilization of the DF3 transcripts. Taken together, these findings indicate that DF3 gene expression is controlled at a transcriptional level in TPA‐

ISSN:0021-9541    

DOI:10.1002/jcp.1041430205

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

年代:1990

数据来源: WILEY

摘要:

AbstractTo elucidate the transmembrane signalling processes initiated by fibroblast growth factor (FGF), we have studied the effect of recombinant basic FGF (bFGF) on various early events associated with mitogenesis in Swiss 3T3 fibroblasts. bFGF, at mitogenic concentrations, neither induced Ca2+mobilization from intracellular stores nor increased the accumulation of inositol phosphates. In contrast, bFGF stimulated the phosphorylation of the Mr 80,000 (80K) cellular protein which is a major substrate of protein kinase C. This effect was potentiated by the diacylglycerol kinase inhibitor R59022. Two‐dimensional polyacrylamide gel electrophoresis and phosphopeptide mapping showed that the 80K phosphoproteins generated in response to bFGF, bombesin, and phorbol 12,13‐dibutyrate were indistinguishable. Down‐regulation of protein kinase C prevented bFGF stimulation of 80K phosphorylation. Other protein kinase C‐dependent early events such as transmodulation of the epidermal growth factor receptor, cytoplasmic alkalinization, inhibition of vasopressin induced increase in cytosolic [Ca2+], and enhancement of cAMP accumulation in response to forskolin were also induced by bFGF. Similar results were obtained when bFGF was added to quiescent cultures of tertiary mouse embryo fibroblasts. We conclude that bFGF stimulates protein kinase C through a signal transduction pathway distinct from inositol phospholipid turnover and Ca2+mobil

ISSN:0021-9541    

DOI:10.1002/jcp.1041430206

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

年代:1990

数据来源: WILEY

摘要:

AbstractWe investigated the roles of second messengers in K‐562 cell differentiation induced by either commitment‐inducing agents (Ara‐C, thymidine), or a noncom‐mitment‐inducing agent (hemin). Cell differentiation induced by both types of agents was inhibited by dbc‐AMP, staurosporine, and H‐7. In contrast, OAG enhanced hemin‐induced cell differentiation, but it inhibited that due to Ara‐C or thymidine. When K‐562 cells were incubated with 4 x 10−6M hemin or 2 x 10−7M Ara‐C for 2 days, an increase of ϵ‐mRNA occurred. The addition of cycloheximide (1 μg/ml) completely blocked this change, suggesting that de novo protein synthesis was necessary for the increase of ϵ‐mRNA. Simultaneous treatment with Ara‐C and cycloheximide for 2 days did not block either the increase of ϵ‐mRNA or that of benzidine‐positive cells, which were measured after 5 days of further incubation without additives. This suggested that the process of Ara‐C‐induced K‐562 cell differentiation could be divided into two steps, i.e., a commitment step and a phenotypic expression step, and that the commitment step was at least partly resistant to cycloheximide. We investigated the roles of second messengers in each step. Our results suggested that PKC may act as a negative regulator of commitment step and as a positive regulator of the phenotypic expression. This may explain the differing effects of OAG on hemin

ISSN:0021-9541    

DOI:10.1002/jcp.1041430207

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

年代:1990

数据来源: WILEY

摘要:

AbstractRibonucleotide reductase activity in S49 T lymphoma cells is cell cycle regulated by de novo protein synthesis of the M2 subunit. There is maximal enzyme activity in S and G2/M phase with low activity and low concentrations of the M2 subunit in G1 phase. Pharmacologic concentrations of cyclic AMP arrest S49 cells in the G1 phase of the cell cycle. We investigated the effect of cyclic AMP on M2 messenger RNA concentrations using RNA from exponentially growing and elu‐triated, cell cycle‐enriched populations. To discern whether cyclic AMP‐induced G1 arrest was associated with low concentrations of M2‐specific messenger RNA, we probed blots with a full‐length cDNA for M2. Cell cycle variation in M2 messenger RNA concentrations was similar in wild‐type, hydroxyurea‐resistant cells with amplified M2 activity, and cyclic AMP‐dependent protein kinase‐deficient cell lines. All lines had low amounts of M2‐specific mRNA in early G1, an increase at the late G1/early S phase interface, a decrease in mid S phase, and another increase in late S phase that continued through G2/M. These concentrations did not directly correlate with enzyme activity, suggesting other regulatory effects might participate in determining ribonucleotide rectase activity. Cyclic AMP exposure appeared to induce cell cycle arrest in early G1 with low M2‐specific messenger RNA concentration. This effect reversed upon washout of the cyclic AMP and was dependent on functional cyclic AMP‐dependent protein kinase (PKA). These results suggest that cyclic AMP arrests S49 mouse T lymphoma cells in early G1 prior to transcriptional ac

ISSN:0021-9541    

DOI:10.1002/jcp.1041430208

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe effect of changes in muscle length on post‐tetanic isometric twitch tension potentiation and myosin P‐light chain phosphorylation‐was studied at 23°C in the mouse extensor digitorum longus muscle. The length‐tension relationship was determined for the same muscles after a 30 min period of quiescence and between 30 s and 3 min after a 1.5 s tetanus at L0. Isometric twitch tension is increased at all muscle lengths after the tetanus; however, the fractional increase in twitch tension rises from 0.2 at L0to a maximum of 0.3 at 1.2 L0.The fractional increase in twitch tension measured at any fixed muscle length is constant between 30 s and 3 min post‐tetanus. P‐light chain phosphorylation remains constant between 30 s and 3 min post‐tetanus followed by a slow decline to basal values. Under fixed length conditions, there is linear relationship between the relative magnitude of the twitch tension and the extent of P‐light chain phosphor‐ylation. Net myosin phosphorylalion measured after a 1.5 s tetanus at 1.23 L0is 35% less than that obtained under the same conditions at L0.Thus, contraction‐induced phosphorylation of P‐light chain decreases with increased muscle length and post‐tetanic potentiation at a constant level of P‐light chain phosphorylation increases with increasing muscle length. These observations may be consistent with alterations in the sarcoplasmic Ca2+ion transient a

ISSN:0021-9541    

DOI:10.1002/jcp.1041430209

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

年代:1990

数据来源: WILEY

摘要:

AbstractClones of F11 hybrid (neuroblastoma X dorsal root neuron) cells have been tested for adherence and neurite outgrowth on three different substrata on which the parental cells display some competence–;plasma fibronectin (pFN) with its multiple receptors, cholera toxin subunit B(CTB) as a model ganglioside GM1‐binding substratum, and platelet factor‐4 (PF4) as a model proteoglycan‐binding substratum. This paradigm tests for independently segregating and overlapping mechanisms of neuritogenesis via transmembrane processes in pluripotent hybrid cells based on random loss of chromosomes contributed by the parent neural cells. For the nine clones tested, attachment was significantly lower on CTB but much higher on PF4 for all clones when compared to their attachment on pFN. Supplementation of cells with GM1 stimulated attachment of only two clones (on all three substrata). Neurite outgrowth was observed in a substratum‐specific pattern and varied from 0 to>60% on pFN; on CTB and PF4 substrata, the patterns were similar to each other but differed markedly from the pattern on pFN. In contrast, PF4‐ and CTB‐directed neurites differed morphologically from each other while sharing some characteristics with neurites on pFN. Supplementation with GM1 or GT1b, but not GD1a, was inhibitory for neurite outgrowth in certain clones. Cycloheximide pretreatment distinguished several classes of clones based on inhibition of neuritogenesis. While most clones on pFN were unaffected, all clones on CTB and PF4 displayed significant and comparable degrees of inhibitior, suggesting the sharing of some protein intermediate(s) on these substrata. Exposure to cycloheximide only during the active period of neuritogenesis generated higher percentages and longer neuritesfor all clones, indicating a widelyused negative regulation mechanism. Based on substratum type and cycloheximide protocols, these data have resolved six or more different transmembrane signalling processes for generating different classes of neurites. Some mechanisms have been segregated into individual clones while others overlap in other clones, providing cell systems for biochemical and molecular biological dissection of th

ISSN:0021-9541    

DOI:10.1002/jcp.1041430210

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

年代:1990

数据来源: WILEY

摘要:

AbstractKeratinocytes from rat skin were separated according to their size in a specially designed unit‐gravity sedimentation chamber. The fractions obtained with this technique showed clear morphological differences, and analysis of size distribution confirmed that size was the criterion for separation. Simultaneous DNA and RNA staining of the fractions with acridine orange and subsequent flow cytometric analysis enabled one to classify cells into resting, proliferating, and differentiating stages. Cell size was not directly correlated with proliferation in situ as determined with acridine orange flow cytometry, nor with proliferative capacity in culture as assayed by BrdU/Hoechst flow cytometry. The smallest cells, exhibiting low DNA and RNA content, which do not proliferate in vivo, required a prolonged period of serum stimulation in vitro to initiate RNA and DNA synthesis. Cells of intermediate size exhibited early RNA synthesis and maximal proliferative capacity, whereas the largest cell population displayed no RNA synthesis in culture and the least proliferative capacity. In conclusion, these results suggest that RNA synthesis early after serum stimulation, in addition to a specific, optimal cell size, correlates with the proliferative capacity of keratinocytes in cell cultur

ISSN:0021-9541    

DOI:10.1002/jcp.1041430211

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

年代:1990

数据来源: WILEY