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Biomedical subjects

R E Scott

Publications and source records attributed to R E Scott.

At least 91 records · Page 5Linked to original sources
PubMed1986

Differentiation, dedifferentiation, and transdifferentiation of BALB/c 3T3 T mesenchymal stem cells: potential significance in metaplasia and neoplasia.

The expression of defects in the control of cellular differentiation is thought to be of etiological significance in the early stages of carcinogenesis. This possibility is supported by a variety of experimental studies including those that have established that metaplastic changes in cells can represent preneoplastic lesions in vivo. To evaluate this question in greater detail, we have used 3T3 T mesenchymal stem cells as a model system. These cells express certain characteristics of preneoplastic cells even though they can regulate their proliferation and even though they can undergo nonterminal and terminal differentiation into adipocytes. For example, they are immortal and aneuploid, and they show a proclivity to undergo spontaneous or induced neoplastic transformation compared to normal human cells. The question we sought to answer in the current experiments concerns whether predifferentiation growth arrest and/or nonterminal differentiation in such preneoplastic cells is completely reversible or whether these processes induce the expression of the new stable program that limits the cells' proliferative potential and reduces the cells' subsequent differentiation potential in a manner comparable to that which is thought to occur in normal stem cells. The results show that arrest at both the predifferentiation state and at the nonterminal differentiation state is a completely reversible phenomenon that does not limit the cells' subsequent growth or differentiation potential. In fact, the results show that, when nonterminally differentiated 3T3 T adipocytes are induced to dedifferentiate, they can subsequently redifferentiate into macrophages. We therefore suggest that preneoplasia as expressed in 3T3 T mesenchymal stem cells is associated with the expression of defects in the ability to integrally control cellular differentiation and proliferation. As a result, the data suggest that such cells express an increased proclivity to undergo metaplastic change and complete neoplastic transformation.

Adipose Tissue↗
PubMed1986

Aproliferin--a human plasma protein that induces the irreversible loss of proliferative potential associated with terminal differentiation.

Cellular proliferation is regulated not only by the action of growth factors and growth inhibitors whose effects are reversible but also by factors that induce the irreversible loss of proliferative potential associated with the terminal event in cellular differentiation. The authors have employed 3T3 T mesenchymal stem cells as a model system to study the terminal event in cellular differentiation because in these cells' distinct nonterminal and terminal states of differentiation can be identified and because transition from the nonterminal to the terminal states of differentiation can be induced by human plasma. In this paper is reported the 20,000-fold purification of a component of human plasma that induces the terminal event in differentiation. This factor is shown to have an apparent molecular weight of approximately 45,000 and an isoelectric point of approximately 7.6. It is trypsin-sensitive, acid and heat-labile, and is resistant to treatment with dithiothreitol and alkali. The ability of this human plasma protein to induce the irreversible loss of proliferative potential associated with the terminal event in differentiation serves as the basis for its designation "aproliferin." The data in this paper in addition show that no other pharmacologic or physiologic agents have been identified that can mimic the biologic effect of aproliferin. Therefore, aproliferin appears to be a functionally distinct protein in human plasma.

Adipose Tissue↗
PubMed1985

An initiator of carcinogenesis selectively and stably inhibits stem cell differentiation: a concept that initiation of carcinogenesis involves multiple phases.

A concept of carcinogenesis was recently devised in our laboratory that suggests the development of defects in the control of cell differentiation is associated with an early phase of carcinogenesis. To test this proposal directly, the effects of an initiator of carcinogenesis (i.e., UV irradiation) on proadipocyte stem cell differentiation and proliferation was assayed. In this regard, 3T3 T proadipocytes represent a nontransformed mesenchymal stem cell line that possesses the ability to regulate its differentiation at a distinct state in the G1 phase of the cell cycle as well as the ability to regulate its proliferation at two additional G1 states that are induced by culture in growth factor-deficient or nutrient-deficient microenvironments. The results establish that a low dosage of 254 nm UV irradiation selectively and stably inhibits the differentiation of a high percentage of proadipocyte stem cells without significantly altering their ability to regulate cellular proliferation in growth factor-deficient or nutrient-deficient culture conditions. Differentiation-defective proadipocyte stem cells are demonstrated not to be completely transformed but to show an increased spontaneous transformation rate, as evidenced by the formation of type III foci in high density cell cultures. These data support the role of defects in the control of differentiation in the initiation of carcinogenesis. Other data, however, also suggest that additional cellular defects must be expressed for a cell to be completely initiated. These observations support a concept that the initiation of carcinogenesis involves multiple phases.

Adipose Tissue↗
PubMed1985

Normal and transformed human prokeratinocytes express divergent effects of a tumor promoter on cell cycle-mediated control of proliferation and differentiation.

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), is shown to be a potent inhibitor of growth of normal human prokeratinocytes (HPK) cultured in serum-free medium. More specifically TPA inhibits the clonal growth of low density HPK cultures in a dose-dependent manner and the anti-proliferative effect of TPA is selective in that the inactive phorbol diester, 4-alpha-12,13-phorbol didecanoate, does not exert a similar effect. One-hour pulse exposure of HPK to TPA also has an effect comparable with continuous exposure to TPA; both treatments induce rapid growth arrest. Flow cytofluorometric analysis of DNA content shows that in TPA-treated HPK growth arrest is associated with accumulation of cells in both the G1 and G2/M phases of the cell cycle. Most interestingly, the data establish that the growth arrest of HPK induced by TPA is irreversible in that treated cells lose their colony-forming potential and that such cells are committed to differentiate without further cell cycle progression when placed in differentiation-promoting medium. In contrast, a human squamous carcinoma cell line, designated SCC-25, is insensitive to the anti-proliferative effect of TPA regardless of whether these cells are cultured in either serum-containing or serum-free medium. These data are interpreted to suggest that transformed human epithelial cells SCC-25 are defective in their ability to regulate their proliferation and differentiation by TPA-sensitive cell cycle-dependent mechanisms.

Adult↗
PubMed1985

Defective control of terminal differentiation and its role in carcinogenesis in the 3T3 T proadipocyte stem cell line.

The expression of defects in the control of terminal cellular differentiation has been implicated to be of etiological significance in the pathogenesis of cancer. However, it has not been established whether or not additional defects in the control of cellular differentiation and proliferation are required for the expression of the completely transformed phenotype. We therefore developed four clones of proadipocyte stem cells that express a single specific defect that limits their capacity to undergo the terminal phase in differentiation. The value of these clones is emphasized by the fact that they show no defects in their ability to regulate nonterminal differentiation or proliferation relative to native nontransformed proadipocyte stem cells. Terminal differentiation-defective clones were therefore assayed to determine if they express the completely transformed phenotype. The results show that differentiation-defective stem cells are not tumorigenic in vivo and do not grow in soft agar, which is an in vitro assay for expression of the transformed phenotype by murine mesenchymal cells. These data are interpreted to support the conclusion that the expression of defects in the control of the terminal phase of differentiation per se is not adequate to induce complete neoplastic transformation.

Adipose Tissue↗
PubMed1984

Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analyses, growth kinetics, and cell cycle studies.

The effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24-hour cell generation time with G1, S, G2, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (greater than 30 population doublings) and neonatal prokeratinocytes (greater than 50 population doublings) is associated with senescence and the G1 arrest of noncycling cells.

Age Factors↗
PubMed1984

Topography of protein kinases and phosphoproteins in the plasma membrane of 3T3 cells.

The plasma membrane of 3T3 cells contains at least two different endogenous cyclic AMP-dependent protein kinase systems. One catalyzes the phosphorylation of endogenous protein substrates, i.e., PP24 and PP14, whereas the other catalyzes the phosphorylation of exogenous substrates. In this paper the topography of these cyclic AMP-dependent phosphorylation systems is described. The results show that the kinases which phosphorylate only exogenous substrates are primarily localized to the outer plasma membrane surface whereas the endogenous cyclic AMP-dependent protein kinase and its two endogenous substrates are localized to the cytoplasmic plasma membrane surface. The data also establish that neither the cytoplasmically orientated kinase nor its substrates has a transmembrane orientation even though factors acting on the outer plasma membrane can affect these proteins. This suggests that functional modulation of the cytoplasmically localized cyclic AMP-dependent phosphorylation system can be mediated by a transmembrane regulatory mechanism. The importance of determining the topography of such plasma membrane phosphorylation systems is emphasized by recent studies which show that neoplastic transformation can be mediated at least in part by protein kinases and/or phosphoproteins which are localized on the cytoplasmic surface of the plasma membrane.

Animals↗
PubMed1984

Biological mechanisms for the loss of the differentiated phenotype by non-terminally differentiated adipocytes.

The differentiation of 3T3 T proadipocyte stem cells is controlled at two related yet distinct states in the G1 phase of the cell cycle. They are designated GD and GD'. GD is the G1 state at which cells must growth arrest prior to differentiation, and GD' is the G1 state at which non-terminal differentiation occurs. Cells arrested at the GD and GD' states have distinct characteristics; yet cells at both states can mediate the integrated control of cellular proliferation and differentiation. In this paper we report on studies designed to further characterize the relationship of these two states, specifically to determine whether non-terminally differentiated GD'-arrested cells can be induced to lose the adipocyte phenotype and revert to the GD state. We report that retinoic acid (RA) and methyl isobutyl xanthine (MIX) can induce non-terminally differentiated GD'-arrested cells to lose the adipocyte phenotype without undergoing DNA synthesis. Such cells that have lost the adipocyte phenotype are also shown to remain in the G1 phase of the cell cycle and to reacquire most of the characteristics of GD-arrested cells. Most importantly, they demonstrate the capacity to redifferentiate without DNA synthesis. We therefore conclude that when non-terminally differentiated GD'-arrested cells are induced to lose the adipocyte phenotype they do indeed revert to the GD state and they thereby become more responsive to environmental influences which can further regulate the integrated control of cellular proliferation and differentiation.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Mechanisms for the initiation and promotion of carcinogenesis: a review and a new concept.

Carcinogenesis in humans is a multistage process, and the two major stages have been designated initiation and promotion. Although the biochemical basis for initiation and promotion remains to be established, recent research has provided important insights into potentially significant biologic mechanisms. These data are reviewed, and a new concept of carcinogenesis is presented. This concept suggests that the initiation of carcinogenesis may result from cellular immortalization and the development of defects in the integrated control of stem cell proliferation and differentiation and that the promotion of carcinogenesis may result when such initiated stem cells develop aberrant autoregulatory growth-control properties.

Animals↗
PubMed1984

Multiplicative interaction between angiotensin II and K concentration in stimulation of aldosterone.

The interaction of angiotensin II and plasma K concentration in stimulating aldosterone secretion was studied in a group of six dogs by determining the aldosterone response to three levels of angiotensin II while the dogs were maintained on three levels of K intake. The levels of angiotensin were 1) the endogenous level, 2) the concentration resulting from infusion of 5 ng X kg-1 X min-1, and 3) the concentration resulting from infusion of 10 ng X kg-1 X min-1. Each level was maintained for 5 days. The three rates of K intake were 10, 100, and 200 meq/day, each maintained for 3 wk. Data were analyzed from days 1, 2, and 5 of the angiotensin infusion periods. The regressions obtained from plotting plasma K (PK) versus aldosterone concentration on day 5 of each level of infusion were 1) for no infusion (endogenous PRA = 0.4 to 0.5 ng ANG I X ml-1 X h-1), aldosterone = 5.04 X PK - 16.56; 2) for 5 ng X kg-1 X min-1 infusion, aldosterone = 12.20 X PK - 39.09; and 3) for 10 ng X kg-1 X min-1 infusion, aldosterone = 35.50 X Pk - 119.31. Each regression was significantly different (P less than 0.001) from the other two. The plasma K axis intercepts, which are the points at which aldosterone secretion is zero, were 3.29, 3.20, and 3.36 for the regression from the 0, 5, and 10 ng X kg-1 X min-1 infusion rates, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone↗
PubMed1984

Safety belt use, ejection and entrapment.

One in every five occupants thrown from a car receives fatal injuries. A motorist who uses a safety belt, in all probability, will not be thrown from the car during a crash. The rate of fatal injury for ejected occupants was found to be 40 times the rate for occupants not thrown from their cars, as determined from national accident sampling data. These data refute the popular notion that "being thrown clear" has survival benefit. In addition, there was no evidence that wearing a safety belt increased fatality risk from vehicle fire or submersion.

Accidents, Traffic↗
PubMed1984

Evidence for secretion of human eosinophil granule major basic protein and Charcot-Leyden crystal protein during eosinophil maturation.

Prior electron microscopic studies have suggested that immature eosinophils degranulate during normal maturation in human bone marrow. This hypothesis was tested by measuring levels of eosinophil granule major basic protein (MBP) and Charcot-Leyden crystal (CLC) protein in bone marrow sinusoidal blood. MBP and CLC protein levels were elevated initially in bone marrow sinusoidal blood from normal volunteers when compared with peripheral blood, and levels of both proteins decreased during serial sampling; CLC protein levels remained significantly elevated, while MBP levels declined to equal those of peripheral blood. To investigate whether MBP and CLC protein were secreted during eosinophil maturation, bone marrow cells were cultured in soft agar; MBP and CLC protein levels were measured in culture supernatants by RIA. There was a significant positive correlation between eosinophil colony growth and levels of each protein. Electron micrographs of cells in soft agar colonies provided ultrastructural evidence for secretion of granule products by immature eosinophils. These results support prior observations that eosinophil promyelocytes degranulate during maturation.

Adult↗
PubMed1983

Cell cycle and cyclic AMP-dependent phosphorylation of plasma membrane proteins p14 and p24: defects in smooth surface transformed cells.

Two proteins which are localized to the cytoplasmic surface of the plasma membrane, p14 and p24, undergo cyclic AMP-dependent phosphorylation in rapidly growing nontransformed murine embryo cells. In this cell system, growth arrest in the G1 phase of the cell cycle induced by growth factor deprivation is associated with the reversible loss in ability to phosphorylate these substrates. By contrast, Simian virus 40 and methylcholanthrene transformed cells show both defective G1 growth control and defects in their ability to phosphorylate p14 and p24 under all tested growth conditions. These data suggested a correlation between defects in the physophorylation of p14 and p24 and defects in the ability of transformed cells to G1 growth arrest. The results of the current studies by contrast show that 3T3 T proadipocytes which have been transformed by the smooth surface tumorigenesis method show different characteristics. They retain the ability to G1 growth arrest in serum-deficient medium. They show cyclic AMP-dependent phosphorylation of p14 and p24 during exponential growth. They do not, however, down regulate p14 and p24 phosphorylation in association with G1 growth arrest. These observations suggest that neoplastic transformation is not necessarily associated with absolute defects in the ability to phosphorylate p14 and p24. Rather, the results of the current study suggest that the inability to modulate the cyclic AMP-dependent phosphorylation of plasma membrane p14 and p24 proteins during the G1 phase of the cell cycle may be more tightly associated with neoplastic transformation.

Adipose Tissue↗
PubMed1983

Efficient differentiation of proadipocyte stem cells on nonadherent surfaces: evidence for differentiation without DNA synthesis.

The differentiation of low density BALB/3T3 T proadipocytes that are cultured in standard tissue culture flasks can be induced by heparinized medium containing human plasma. It has been shown that under these conditions, cells first growth-arrest at a distinct state in the G1 phase of the cell cycle, designated GD, and thereafter differentiate within 8 to 12 days. In this paper, we report that the kinetics of proadipocyte differentiation can be significantly accelerated by culture of cells in differentiation-promoting medium on non-adherent surfaces, such as agarose-coated plates or bacteriological Petri dishes. Data also show that in a nonadherent microenvironment extensive differentiation can occur in the absence of DNA synthesis. This was established most convincingly by the demonstration that placement of mitotic cells in heparinized medium containing human plasma and hydroxyurea on agarose-coated Petri dishes induced 70-80% of the cells to GD arrest and differentiate without traversing the S phase of the cell cycle. It is concluded that under appropriate microenvironmental conditions metabolic events that occur solely in the late M or early G1 phase of the cell cycle can mediate the integrated control of proadipocyte proliferation and differentiation.

Adipose Tissue↗
PubMed1983

Differential mitogenic effects of methyl isobutyl xanthine and a tumor growth factor on G1-arrested 3T3 T proadipocytes at the predifferentiation GD state and the growth-factor deficiency GS state.

Two growth-states exist in the G1 phase of the 3T3 T proadipocyte cell cycle. GD is the arrest state at which proadipocytes must growth-arrest prior to differentiation. GS is the arrest state at which proadipocytes growth-arrest following deprivation of serum or growth factors. In an attempt to further distinguish these arrest states, we have compared the relative ability of a variety of mitogens to induce GD- and GS-arrested cells to initiate DNA synthesis. The data show that GD-arrested cells at both high and low densities can be induced to proliferate by methyl-isobutyl-xanthine (MIX), whereas high and low density GS-arrested cells are not. The data also show that a tumor growth factor can stimulate the proliferation of both high and low density GD- and GS-arrested cells, whereas other agents are poor mitogens for high density GD-arrested cells. We conclude that MIX and a tumor growth factor (TUGF) can serve as probes to study the characteristics of the GD arrest state.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Cell surface characteristics of proadipocytes growth arrested at the predifferentiation GD state. Defects associated with neoplastic transformation.

The differentiation of murine proadipocytes is preceded by growth arrest at a specific state in the G1 phase of the cell cycle, designated GD. The GD arrest state has been shown to be distinct from other G1 arrest states, including those induced by serum or growth factor deprivation, designated GS, and by nutrient deprivation, designated GN. Defects in the control of growth arrest at GD have also been correlated with carcinogenesis. In this study we have analyzed the cell surface characteristics of nontransformed proadipocytes at various states in the G1 phase of the cell cycle by scanning electron microscopy. The results show that abundant cell surface microvilli, 2.0 to 4.0 micrometer long and 0.2 to 0.4 micrometer in diameter, develop when proadipocytes are cultured in medium that induces GD arrest but not when they are cultured under conditions that induce GS or GN arrest. The results also show that initiated and transformed proadipocytes fail to develop prominent cell surface microvilli when cultured in differentiation-promoting medium; they also fail to GD arrest and differentiate.

Adipose Tissue↗