Biomedical subjects

R E Scott

Publications and source records attributed to R E Scott.

At least 37 records · Page 2Linked to original sources

PubMed1994

The developmental expression in rat of proteases furin, PC1, PC2, and carboxypeptidase E: implications for early maturation of proteolytic processing capacity.

The genes encoding mammalian subtilisin-like endoproteases furin, PC1, and PC2 have been isolated and are implicated in endoproteolytic cleavage of precursor molecules, which is a key step in posttranslational maturation of proproteins and neuropeptide precursors. Following endoproteolytic cleavage, the carboxyl-terminal basic amino acid residues are removed by carboxypeptidase E (CPE). We have examined the expression of these genes during rat development by in situ hybridization and compared their expression patterns to those of potential substrates. In the primitive streak stage of embryogenesis (e7) furin is expressed in both endoderm and mesoderm. This overall expression pattern is maintained until e10, when a distinctly higher level of furin expression is observed in the heart and liver primordia. In mid- and late gestational stages furin is broadly expressed in the peripheral tissues, and, therefore, may contribute to the proteolytic processing of numerous fetal proproteins, such as the precursors for natriuretic factors in heart and IGF-II throughout the embryo. In contrast, the expressions of PC1 and PC2 are initiated much later (e13) and are mainly confined to the developing nervous system, but with distinct spatial distributions. At midgestational ages, PC1 mRNA is mainly expressed in the hypothalamus and peripheral ganglia, while PC2 is expressed not only in these tissues but also in the thalamus, midbrain, pons, medulla oblongata, cortical plate, and spinal cord. Besides neuropeptide precursor processing in the nervous system, PC1 and PC2 may also be involved in the proteolytic processing in additional regions as evidenced by the finding that both PC1 and PC2 mRNAs are expressed in the embryonic pituitary and pancreas. CPE mRNA is expressed in both neural tissues and some non-neural tissues. In the developing nervous system, the expression of CPE encompasses all the regions where PC1 and PC2 are expressed and in fact includes most brain regions as neurogenesis proceeds. CPE mRNA is also expressed in some peripheral tissues, such as the embryonic heart and cartilage primordia, and in some cases its expression overlaps with furin expression. Thus, CPE may functionally collaborate during development with the subtilisin family of endoproteases in the completion of proteolytic processing of neuropeptide precursors in the nervous system and proproteins in the peripheral tissues. In the pituitary, the endoproteolytic processing of polyfunctional precursor proopiomelanocortin (POMC) occurs in a developmentally regulated manner. We have shown that while PC2 mRNA is predominantly expressed in the intermediate lobe in the adult, we observed an increased expression of PC2 mRNA in developing rat anterior lobe, peaking at early postnatal stages.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Increased NAD(P)H:(quinone-acceptor)oxidoreductase activity is associated with density-dependent growth inhibition of normal but not transformed cells.

The activity of DT-diaphorase [NAD(P)H:(quinone-acceptor)oxidoreductase] is increased 7-fold in wild-type BALB/c 3T3T cells as they reach confluence and become density growth arrested. Harvesting and replating the cells at low density resulted in a loss of DT-diaphorase with a half time of 7 h, and removal of serum from high-density growth-arrested cells resulted in a decrease in DT-diaphorase with a half time of 3 days. Platelet-derived growth factor and insulin together, but not singly, maintain elevated DT-diaphorase levels in high-density growth-arrested BALB/c 3T3T cells. The increase in DT-diaphorase at high density diminished proportionately to the extent of transformation in four cell lines, 4NQO-3T3T, UV-3T3T, EJras-3T3T. and CSV3-1-3T3T. The most transformed cell line, CSV3-1-3T3T, showed no increase in DT-diaphorase at high density. Since there was no increase in DT-diaphorase mRNA in high-density growth-arrested wild-type BALB/c 3T3T cells compared to rapidly growing cells, the increase in DT-diaphorase activity at high density is most likely due to posttranslational events. High-density growth-arrested wild-type BALB/c 3T3 cells exhibited a greater sensitivity to growth inhibition by the antitumor quinone diaziquone [1,4-cyclohexadiene-1,4- dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ether], which is metabolically activated by DT-diaphorase, than do low-cell-density, growth-arrested cells. The significance of the increase in DT-diaphorase at high cell density in normal cells and its loss in transformed cells may be related to the phenomenon of density-dependent growth inhibition in nontransformed but not in transformed cells.

3T3 Cells↗

PubMed1993

Induction of SV40 early transcription by type I interferon.

Interferons (IFN) have cancer suppressor activities for many transformed cells; however, IFN does not suppress transformation by SV40 large T antigen. The studies described in this paper therefore evaluated the effect of IFN on SV40-promoted gene expression in 3T3T cells. The results show that SV40-promoted gene expression can be induced 200% or three-fold by Type I IFN treatment regardless of whether beta-galactosidase or chloramphenicol acetyltransferase (CAT) is used as the reporter gene. This IFN effect is dosage-dependent, requires 24-48 h of exposure for maximum induction of CAT activity, and is probably not due to a post-transcriptional effect of IFN on CAT because IFN has no effect on CAT expression driven by thymidine kinase promoter. The induction of SV40 early transcription by IFN does, however, require that the integration of plasmid within the cell's genome. Additional data specifically show that the SV40 promoter is required for IFN's effect because IFN will not induce CAT if the SV40 enhancer is inserted upstream of thymidine kinase promoter to control expression of CAT gene.

3T3 Cells↗

PubMed1993

Inhibition of distinct steps in the adipocyte differentiation pathway in 3T3 T mesenchymal stem cells by dimethyl sulphoxide (DMSO).

The process of adipocyte differentiation in murine 3T3 T mesenchymal stem cells involves three well-defined steps: 1 predifferentiation growth arrest; 2 non-terminal (reversible) differentiation and 3 terminal differentiation associated with the irreversible loss of proliferative potential. To further investigate these processes, the effects of dimethyl sulphoxide (DMSO), an agent that affects differentiation in several other cell systems, was tested. The results show that DMSO modulates two distinct steps of adipocyte differentiation. The first effect is evident when growing 3T3 T cells are cultured in differentiation-inducing medium in the presence of DMSO. Therein the expression of adipocyte phenotype is inhibited because the cells fail to growth-arrest at the predifferentiation growth arrest state. Instead in the presence of DMSO, cells growth-arrest at a biological state that does not support differentiation. The second effect is evident if nonterminally differentiated adipocytes are cultured in terminal differentiation-inducing medium containing DMSO. Therein the terminal step in differentiation is inhibited. These inhibitory effects occur in a dosage-dependent manner; maximum inhibition of differentiation requires 2% DMSO. Therefore, whereas DMSO typically promotes differentiation in other cell systems, DMSO inhibits multiple steps in the process of adipocyte differentiation. These observations support the conclusion that a single pharmacological agent can have markedly different effects on specific cell types. Even more important, the data establish that DMSO can now be used as a tool to study the molecular mechanisms involved in the multistep process of adipocyte differentiation.

3T3 Cells↗

PubMed1993

Decreased expression of protooncogenes c-fos, c-myc, and c-jun following polyamine depletion in IEC-6 cells.

Direct exposure of small intestinal mucosal cells to luminal polyamines stimulates proliferation. This study tests the hypothesis that the protooncogenes c-fos, c-myc, c-jun, and junB are involved in the mechanism by which polyamines modulate mucosal growth. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence of absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme for polyamine synthesis. Cellular polyamine levels, cell growth, and relative abundance of c-fos, c-myc, c-jun, and junB mRNAs, were measured at 1, 2, 4, 6, 8, and 12 days after initial plating. The intracellular polyamines, spermidine and spermine, and their precursor, putrescine, in DFMO-treated cells decreased significantly at 2 days and remained depleted thereafter. Although DFMO profoundly decreased growth and final cell number, both control and DFMO-treated cells entered a plateau phase by 6 days. In control cells, c-myc and c-jun mRNA levels significantly increased on days 4-6 and then returned to a basal level of expression, which was maintained thereafter. c-fos mRNA in quiescent cells after 24 h serum deprivation was significantly stimulated by 5% dFBS, although a steady-state level of c-fos mRNA was undetectable in control cells. Treatment with DFMO not only prevented increased expression of c-myc and c-jun protooncogenes at 4 days, but also significantly reduced steady-state levels of c-myc and c-jun mRNA between 6 and 12 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Developmental regulation of proopiomelanocortin gene expression in the fetal and neonatal rat pituitary.

Expression of the POMC gene and secretion of its peptide products are under complex regulation in the pituitary by multiple factors. CRF stimulates POMC transcription and secretion in both adult anterior (AL) and intermediate (IL) pituitary lobes, whereas glucocorticoids have an inhibitory effect on POMC in the AL, but little, if any, effect in the IL. To determine when transcriptional responses elicited by these factors begin during development and whether they undergo changes during ontogeny, we used a solution hybridization/nuclease protection assay with a POMC exon 1-intron A splice junction probe to analyze simultaneously the levels of intron A-containing POMC heterogeneous nuclear RNA (hnRNA) and POMC mRNA in explant fetal and neonatal rat pituitaries. We examined responses to 8-bromo-cAMP, CRF, and dexamethasone (dex) at stages before and after innervation of the IL by dopaminergic neurons from the hypothalamus. Treatment of embryonic day 15 (e15) whole pituitaries with CRF (10(-7) M) for 1 h led to a 2.5-fold increase in the level of POMC hnRNA, while pretreatment with dex (10(-6) M) inhibited the CRF-induced stimulation of POMC transcription. These results demonstrate that by e15, POMC transcription is already responsive to both CRF and dex, and thus, functional receptors (coupled effectively to the POMC promoter) are present by this age. Initial studies of POMC mRNA levels at early postnatal ages showed that 1 mM 8-bromo-cAMP stimulated postnatal day 1 (p1) and p10 AL and neurointermediate lobe (NIL) POMC mRNA levels, and 10(-6) M dex inhibited this stimulation in p1 AL, p10 AL, and p1 NIL, but not in p10 NIL. These studies were extended to examine POMC hnRNA responses at these ages. Treatment with CRF for 1 h increased POMC hnRNA 1.9- and 1.5-fold in p1 and p10 AL, respectively, and pretreatment with dex blocked these CRF-mediated effects on AL POMC transcription. In the NIL on p1, CRF induced a 2-fold increase in POMC hnRNA, which (like that in the AL) was inhibited by 30-min pretreatment with dex; in contrast, on p10, dex did not affect the CRF-induced increase in POMC hnRNA. The glucocorticoid receptor subtype responsible for this effect was identified using treatments with specific agonist and antagonists. The type II receptor agonist RU 28362 had an effect similar to that of dex; at both 10(-6) and 10(-8) M, RU 28362 inhibited CRF-induced increases in POMC hnRNA in p1 NIL and p1 and p10 AL.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1993

Regulation of differentiation, proliferation and cancer suppressor activity.

This review suggests that carcinogenesis is linked to defects in linkages between cellular differentiation and regulation of cell proliferation. These linkages are discussed in terms of terminal and nonterminal states of differentiation and their relationship to the control of proliferation. The ability of differentiation to regulate proliferation potential proteins and cancer suppressor genes is also discussed, because these mechanisms may be important for cancer prevention and therapy.

Animals↗

PubMed1992

Development and characterization of a rapidly proliferating, well-differentiated cell line derived from normal adult human osteoblast-like cells transfected with SV40 large T antigen.

A new bone cell line was established by transfecting normal adult human osteoblast-like (hOB) cells, derived from a 68-year-old woman, with the plasmid pSV3 neo. The plasmid included coding sequences and promotors for the large and small T antigens of the SV40 virus as well as resistance to the antibiotics neomycin and G418. A single antibiotic-resistant colony was located and cloned. Large tumor antigen production in the clonal cell line was confirmed by indirect immunofluorescence study. Treatment with 1,25-dihydroxy-vitamin D3 increased steady-state concentrations of protein and mRNA for osteocalcin and for alkaline phosphatase. Northern blot analyses also demonstrated the presence of mRNAs for alpha(I)-procollagen, osteopontin 1a, transforming growth factor beta, and interleukin-1 beta. The plasma membrane calcium pump and osteonectin were identified by immunocytochemical analysis. These cells produced a matrix that mineralized when beta-glycerophosphate was added to their cultures. As assessed by functional receptor assays, both estrogen and androgen receptors were present and functional, although at low concentrations. Treatment with parathyroid hormone did not stimulate adenylate cyclase activity. Thus, these cells are a well-differentiated, steroid-responsive clonal cell line that closely approximates the phenotype of the mature osteoblast. They should serve as an excellent model for the study of osteoblast biology.

Aged↗

PubMed1992

Repression of SV40 T oncoprotein expression by DMSO.

SV40 large T oncoprotein-transformed murine mesenchymal 3T3 T stem cells (CSV3 cells) can be induced to growth arrest and then differentiate into adipocytes. When differentiation occurs, SV40 T oncoprotein expression is repressed (Estervig et al., J Virol 63:2718, 1989). To determine if repression of T oncoprotein expression can also be induced pharmacologically, the effect of a variety of agents that have been reported to effect differentiation in various cell types but not in 3T3 T or CSV3 cells was tested. This rationale suggests that if any of these agents repress T oncoprotein expression in CSV3 cells, then the results would establish that repression of T oncoprotein expression can be mediated by mechanisms independent of overt differentiation. The results show that dimethylsulfoxide (DMSO) is the only agent tested that represses T oncoprotein expression in CSV3 cells. Repression occurs in a dosage-dependent manner within 24-96 hours after exposure to DMSO. The effect of DMSO on T oncoprotein expression is mediated by posttranslational mechanisms that decrease the stability of the T oncoprotein. DMSO-induced repression of T oncoprotein expression is also associated with reversion of the transformed phenotype in CSV3 cells as demonstrated by the loss of responsiveness to a specific transformation-associated mitogen. These data support the conclusion that the pharmacological repression of T oncoprotein expression represents a form of cancer suppressor activity that can be mediated by a distinct molecular mechanism.

Acetamides↗

PubMed1992

Induction of c-jun independent of PKC, pertussis toxin-sensitive G protein, and polyamines in quiescent SV40-transformed 3T3 T cells.

CSV3 clones of simian virus 40 large T antigen-transformed murine 3T3 T cells can be made quiescent as part of a differentiation process. In these quiescent cells, insulin- and vanadate-induced mitogenesis are both associated with the induction of the c-jun proto-oncogene (Wang and Scott 1991 J. Cell. Physiol. 147, 102-110; Wang et al. 1991 Cell Growth Differ. 2, 645-652). The current studies were therefore designed to compare the early signal transduction pathways employed by insulin and vanadate to regulate c-jun expression. In quiescent CSV3-1 cells, down-regulation of protein kinase C by prolonged exposure to 12-O-tetra-decanoylphorbol-13-acetate or inhibition of protein kinase C activity by treatment with the protein kinase C antagonist staurosporine is shown not to affect c-jun induction by insulin or vanadate. This suggests that both insulin and vanadate act in a protein kinase C-independent manner. Insulin's effect on c-jun induction does, however, involve a G protein because insulin's effect can be inhibited by pertussis toxin. In contrast, vanadate induction of c-jun is not affected by pertussis toxin. Genistein, a general tyrosine kinase inhibitor, can inhibit the ability of vanadate to induce c-jun but it does not inhibit insulin's effect. Finally, the depletion of polyamines, particularly spermidine, by DL-alpha-difluoromethylornithine treatment also prevents c-jun induction by insulin but DL-alpha-difluoromethylornithine treatment has no effect on c-jun induction by vanadate. These observations indicate that the c-jun induction by insulin and vanadate in CSV3-1 cells is mediated by different signal transduction mechanisms. Together with our previously published data, these results suggest that c-jun can be induced independent of protein kinase C activation, without involvement of pertussis toxin-sensitive G protein, independent of induction of c-fos, and without expression of high levels of intracellular polyamines.

3T3 Cells↗

PubMed1992

Stable induction of c-jun mRNA expression in normal human keratinocytes by agents that induce predifferentiation growth arrest.

A variety of agents can induce predifferentiation growth arrest (PGA) in human keratinocytes; these include transforming growth factor beta 1 (TGF-beta 1) and razoxane. We evaluated the ability of these and other agents to induce the expression of a variety of transcription factor genes including c-fos, c-myc, junB, and c-jun. The results show that both TGF-beta 1 and razoxane induce maximal c-jun mRNA expression 4 days after initiation of treatment concurrent with the development of PGA. In contrast, no detectable induction of c-fos, c-myc, or junB was observed. Keratinocytes maintained in the presence of TGF-beta 1 for an additional 3 days continued to show high levels of c-jun mRNA, indicating stable induction. Razoxane treatment also induces PGA and high c-jun mRNA levels for 4 days, but thereafter a decay of c-jun expression occurs. Run-off transcription experiments comparing rapidly growing cells with cells treated with TGF-beta 1 for 4 days demonstrated a significant increase in transcriptional activity of the c-jun gene. This result indicates that the increase in c-jun gene expression is due in part to a change in transcriptional regulation of c-jun. The stable induction of c-jun mRNA in keratinocytes at the PGA state is unique because the induction of this gene is usually transient. The finding that c-fos is not coinduced suggests that c-Jun homodimers or other AP-1 heterodimers may be formed at the PGA state to facilitate the stable induction of c-jun mRNA. This experimental system should therefore serve as a model system to study the molecular mechanisms for the stable control of c-jun gene expression and the control of AP-1-dependent gene expression during the process of keratinocyte differentiation.

Cell Differentiation↗

PubMed1992

Biological characteristics of primary cultures of human gallbladder epithelial cells.

Epithelial cells of the gallbladder have potential to represent an important model for studies of ductal epithelial in normal and pathological states. We therefore initiated studies to establish human gallbladder epithelial cells (GBEC) in culture. GBEC were isolated by trypsinization of small tissue fragments from human gallbladders obtained at cholecystectomy; cells were plated on tissue culture dishes and grown in defined MCDB 153 medium containing added growth factors. In this medium, GBEC showed a plating efficiency of approximately 1%; those GBEC that attached formed colonies and proliferated, as demonstrated by autoradiographic analysis of [3H]thymidine incorporation into DNA. Cultured GBEC expressed two markers found on GBEC in situ, i.e., gamma-glutamyl transpeptidase and cytokeratin 19. By using various attachment substrates, with and without added serum, increased plating efficiency and better growth were achieved. When type IV collagen was used as substrate and 10% fetal bovine serum was added to MCDB 153, passage of GBEC was possible, and cells proliferated through five to six population doublings. GBEC in culture under all conditions eventually enlarged, showed vacuolization, and demonstrated irreversible growth arrest. Nonetheless, the culture conditions described here allow for preparation of large quantities of highly enriched human GBEC.

Cell Division↗

PubMed1991

Clinical chemistry.

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Chemistry, Clinical↗

PubMed1991

Lack of a direct effect of estrogen on proliferation and differentiation of normal human osteoblast-like cells.

Although osteoblasts contain estrogen receptors, it is unclear whether estrogen has direct effects on osteoblast proliferation and differentiation. We evaluated the effects of 17 beta-estradiol treatment (1 pM to 10 nM) on the proliferation and differentiation of cultured normal adult human cells that expressed many of the phenotypic characteristics and hormonal sensitivities of mature osteoblasts (hOB cells). Treatment of hOB cells with estradiol for as long as 144 h did not affect the rate of DNA synthesis and had minimal, if any, effects on differentiated function. Whereas alkaline phosphatase activity was increased by nearly twofold (P less than 0.01) when the hOB cells were treated with 1 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], treatment with estradiol had no effect when given alone and did not affect the cells' response to 1,25-(OH)2D3. Similarly, the release of bone gla protein (BGP, osteocalcin) was induced by treatment with 1,25-(OH)2D3 (P less than 0.05), but estradiol treatment did not affect this response. Cellular levels of mRNA for alkaline phosphatase and BGP were not altered by estradiol treatment. We conclude that estradiol treatment does not have major effects on the growth or differentiation of cultured hOB cells. These results are consistent with previous observations in vivo that indicate estrogen acts principally to decrease bone resorption, not to modulate its formation.

Cell Differentiation↗

PubMed1991

Insulin-induced mitogenesis associated with transformation by the SV40 large T antigen.

Simian virus 40 (SV40) large T antigen-transformed cells typically show a markedly reduced serum requirement for growth and the inability to growth arrest and differentiate. An SV40 large T antigen-transformed 3T3 T cell line, CSV3-1, that can growth arrest and differentiate into adipocytes with high efficiency has, however, recently been described (Scott et al: Proc. Natl. Acad. Sci. U.S.A. 86:1652-1656, 1989; Estervig et al: J. Virol. 63:2718-2725, 1989; J. Cell. Physiol. 142:552-558, 1990). The results of the current studies using these cells show that whereas quiescent 3T3 T cells show no mitogenic response to insulin, quiescent CSV3-1 cells show a highly significant insulin-induced mitogenic responsiveness in the absence of other added growth factors. Maximum mitogenesis was observed at an insulin concentration of 1 microgram/ml, which induced 40-70% of the cells to undergo DNA synthesis within 48 hours. The half maximum response was achieved with 1-10 ng/ml of insulin. Insulin's mitogenic effect on CSV3-1 cells was evident under several different culture conditions that induce quiescence and was not mediated by any detectable autocrine growth factors that might make CSV3-1 cells competent to respond to insulin. In CSV3-1 cells insulin appears to act on its own receptor rather than on the IGF-1 receptor, because at comparable dosages IGF-1 is 10- to 100-fold less effective than insulin. Insulin also is shown to be a mitogen for another SV40-transformed cell line, CSV3-35, which can be growth arrested; in contrast insulin has no mitogenic effect on two control cell lines that are stably transfected with pSV2neo, a plasmid containing SV40 early promoter/enhancer but lacking large T antigen gene: These results suggest a significant relationship between SV40 T antigen-associated transformation and the expression of mitogenic responsiveness to insulin.

Adipose Tissue↗

PubMed1991

Selective induction of c-jun and jun-B but not c-fos or c-myc during mitogenesis in SV40-transformed cells at the predifferentiation growth arrest state.

Insulin has recently been reported to function as a complete mitogen for SV40 large T antigen-transformed 3T3 T-cells, designated CSV3-1, but not for nontransformed 3T3 T-cells (H. Wang and R. E. Scott, J. Cell. Physiol., 147: 102-110, 1991). It is now reported that sodium orthovanadate mimics this effect of insulin. For example, when exposed to 1-5 microM vanadate, most predifferentiation growth-arrested CSV3-1 cells undergo DNA synthesis within 24 h, but neither vanadate nor insulin induces mitogenesis in nontransformed 3T3 T-cells. To investigate the possible mechanisms by which mitogenesis is induced in CSV3-1 cells, the effects of insulin and vanadate on the expression of growth-related genes were examined. Whereas insulin and vanadate had no effect on the expression of c-fos, c-myc, c-jun, jun-B, or ornithine decarboxylase activity in nontransformed 3T3 T-cells, insulin and vanadate showed different effects on the expression of these genes in CSV3-1 cells. Insulin induced a rapid and transient accumulation of c-fos mRNA followed by induction of c-myc, c-jun, jun-B, and ornithine decarboxylase. In contrast, vanadate induced the expression of c-jun, jun-B, and ornithine decarboxylase without inducing c-fos and c-myc. These observations suggest that SV40 large T antigen may play an important role in insulin- and vanadate-induced mitogenesis and that insulin and vanadate may mediate their mitogenic effects by different signal transduction pathways.

Antigens, Viral, Tumor↗

PubMed1990

Three distinct effects of SV40 T-antigen gene transfection on cellular differentiation.

SV40 large T-antigen-induced transformation has been reported to block differentiation, but the mechanism(s) of this effect has not been established. The results presented here show that stable transfection of the SV40 T-antigen gene, via the pSV3neo plasmid, has at least three distinct effects on 3T3T adipocyte differentiation. Cells first show a decreased ability to undergo predifferentiation growth arrest, which is a prerequisite for in vitro 3T3T adipocyte differentiation. However, if predifferentiation growth arrest is accomplished by use of stringent differentiation-inducing culture conditions, adipocyte differentiation can occur with high frequency. The pSV3neo-transfected cell clones also show other modifications of the adipocyte differentiation process, including changes in nonterminal (reversible) and terminal (irreversible) steps of adipocyte differentiation. When compared to nontransfected 3T3T cells, the cell clones containing pSV3neo require markedly reduced growth factor concentrations to restimulate proliferation of nonterminally differentiated adipocytes and the terminal step of differentiation is also blocked. These results suggest that integration of the T-antigen gene, through pSV3neo transfection, has multiple effects on the cellular mechanisms of differentiation. It does not block the differentiation process per se; rather it appears to make cells highly sensitive to proliferation signals, thereby making differentiation more difficult.

Adipose Tissue↗

PubMed1990

Specific expression of proteins and phosphoproteins in 3T3 T mesenchymal stem cells at distinct growth arrest and differentiation states.

Murine mesenchymal stem cells can be induced to arrest their growth at a series of growth and differentiation states in the G1 phase of the cell cycle. These include the predifferentiation arrest state (GD) at which the integrated control of proliferation and differentiation is mediated, the growth factor/serum deficiency arrest state (GS), and the nutrient deficiency arrest state (GN). Cells at states of reversible nonterminal differentiation (GD') and irreversible terminal differentiation (TD) can also be isolated. In this paper we have employed 1- and 2-dimensional (D) gel electrophoresis to evaluate changes in specific proteins that occur during the various growth and differentiation states of 3T3 T mesenchymal stem cells. The protein composition of membrane, microsome and cytosol preparations of cells arrested at GD, GS and GN states was determined by 2-D gel electrophoresis. More than 50 distinct polypeptides could be identified for each arrest state in gels analysed by a silver staining procedure or by autoradiography following [35S]-methionine labelling. A second series of studies established that a more limited number of differences could be identified if phosphoproteins were analysed by 1-D gel electrophoresis in cells at the GS, GD, GD' and TD states. These results established that one distinct 37 kD phosphoprotein is present in all growth arrested cells and that two distinct differentiation-associated phosphoproteins with molecular weights of 29 kD and 72 kD are present in cells at the GD' and TD states. Thus, the composition of proteins and phosphoproteins in mesenchymal stem cells serves to characterize different states of growth arrest and differentiation.2+he identification of differential

Animals↗