Growth arrest of proadipocytes at a distinct predifferentiation G1 state associated with cytotoxic responsiveness to 8-bromo cyclic AMP

The control of cell proliferation can result from the coupling of growth arrest and differentiation. In this regard, we recently demonstrated that growth arrest which precedes the differentiation of 3T3 T proadipocytes must occur at a distinct state in the G1 phase of the cell cycle (GD). Cells arrested at GD differ in several biological parameters from cells arrested in G1 at other states induced by either serum deprivation (GS) or nutrient deficiency (GN). Specifically, GD-arrested cells can differentiate in the absence of DNA synthesis and GD-arrested cells can be induced to proliferate when stimulated with 1-methyl-3-isobutylxanthine; GS- and GN-arrested cells cannot. In addition, GD-, GS- and GN-arrested cells reside at topographically distinct states in G1. We now report that GD-arrested proadipocytes are also distinct in that they are highly sensitive to a cytotoxic effect of 8-bromocyclic AMP, whereas GS- and GN-arrested cells are not.     

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.     

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     

Coupling of proadipocyte growth arrest and differentiation. I. Induction by heparinized medium containing human plasma

The differentiation of proadipocytes in vitro typically required prolonged culture of cells as a high density in high concentrations of serum and added hormones. With such culture conditions it is difficult to design experiments to determine the mechanisms that control the differentiation process. We now describe the rapid and parasynchronous growth arrest and differentiation of low density murine proadipocytes in heparinized medium containing only human plasma. When low density cells are cultured under these conditions, growth arrest at a distinct state in the G1 phase of the cell cycle occurs within 2 d and the differentiation of 80-100% of the cell population occurs within 4 d thereafter. The factors in human plasma which promote growth arrest and differentiation are heat labile and can be separated by barium adsorption. In the following paper we have used these methods to show that there are five separate phases which regulate the coupling of proadipocyte growth arrest and differentiation. The data reported in this paper establish that: (a) high cell density and extensive cell-to- cell contact are not required for adipocyte differentiation, (b) prolonged culture is not required for adipocyte differentiation, and (c) high concentrations of serum and/or added hormones are not required for adipocyte differentiation.     

Adipose conversion of 3T3-L1 cells in a serum-free culture system depends on epidermal growth factor, insulin-like growth factor I, corticosterone, and cyclic AMP.

A culture system for 3T3-L1 preadipocytes based on a serum-free chemically defined medium containing fetuin, transferrin, and pantothenate is described. In this system, adipose conversion depends on the following conditions. 1) In the presence of high insulin concentrations (1 microM), addition of corticosterone together with 1-methyl-3-isobutylxanthine (MIX) for not more than the first 4 days after confluence to the culture medium induces maximal adipose conversion within 12-14 days. MIX may be replaced by forskolin or permeable analogues of cAMP, indicating that its effect is due to elevated cellular cAMP levels. 2) At low insulin concentrations (1 nM), adipose conversion is reduced. Growth hormone or insulin-like growth factor I together with epidermal growth factor have to be present as a medium supplement together with corticosterone and MIX to get maximal adipose conversion. 3) The induction of adipose conversion by corticosterone and MIX in the presence of either high insulin concentrations or insulin-like growth factor I together with epidermal growth factor is accompanied by post-confluent mitoses. Inhibitors of DNA replication markedly reduce adipose conversion. Fibroblast growth factor and platelet-derived growth factor, although acting as potent mitogens on 3T3-L1 cells, do not support adipose conversion induced by corticosterone and MIX.     

2-amino-isobutyric acid and 3-O-methyl-D-glucose transport in 3T3, SV 40-transformed 3T3 and revertant cell lines.

In order to further investigate the connection between transport and growth control, 3T3 cells, SV40 transformed 3T3 cells (SV101), and three revertant cell lines derived from SV101 which have regained certain manifestations of growth control were used. Transport rates of 2-amino-isobutyric acid and 3-O-methyl-D-glucose were measured in sparse, confluent, serum-starved, and serum-stimulated cultures. As shown before, cessation of 3T3 cell growth in G0 under conditions of confluence or serum deprivation was associated with reduced rates of transport for both compounds, whereas the density and serum dependence of growth and transport was largely eliminated in SV101. The density revertant F1SV101, which has regained density regulation of growth similar to 3T3 cells, has also regained density regulation of transport. Neither growth nor transport were serum dependent. The serum revertants AgammaSV7 and LsSV6 have regained both density and serum regulation of growth, but not according to the original mechanism of 3T3 cells of entry into a Go state. Transport was high under conditions of confluence or serum deprivation. Thus for these cells rates of transport were not reduced simply as a consequences of slower cell growth nor were low transport rates responsible for growth arrest. The data are consistent with the possibility that growth arrest specifically in the G0 state could shut off a number of cellular activities, including transport.     

Nonterminally differentiated cells express decreased growth factor responsiveness

In 3T3 T mesenchymal stem cells, at least four types of biological states exist that can mediate the control of cell differentiation and/or proliferation. These include the predifferentiation growth arrest state, the nonterminal differentiation state, the terminal differentiation state, and a growth arrest state induced by growth factor/serum deficiency. The current studies were performed to investigate the relative mitogenic responsiveness of cells at these four states and specifically to determine if nonterminally differentiated cells show decreased responsiveness to specific mitogens. Twenty-five different serum, plasma, and growth factor combinations were evaluated. The results show that undifferentiated, growth-arrested cells are highly responsive to numerous mitogens and that by definition terminally differentiated cells are not responsive to any mitogens. In contrast, nonterminally differentiated cells demonstrate a unique pattern of mitogenic responsiveness. Whereas nonterminally differentiated cells can be stimulated to proliferate by high concentrations of serum or plasma supplemented with growth factors, they cannot be stimulated to proliferate by combinations of multiple purified growth factors. These results suggest that the process of nonterminal differentiation is associated with a significant change in factors/cofactors required to stimulate cell proliferation and that these factors/cofactors are present in plasma.     

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

Abstract. The process of adipocyte differentiation in murine 3T3 T mesenchymal stem cells involves three well-defined steps: 1 predifferentiation growth arrest; 2 nonterminal (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 growtharrest 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.     

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 G_D, 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 G_D 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.     

Epidermal growth factor and the control of proliferation of Balb 3T3 and benzo[a]pyrene-transformed Balb 3T3 cells.

Benzo[a]pyrene-transformed Balb 3T3 cells (BP3T3) exhibit "normal" growth controls at low concentrations of serum. Epidermal growth factor (EGF) stimulates DNA synthesis and cell division in both Balb 3T3 and BP3T3 cells at physiological concentrations. The growth response of BP3T3 cells to EGF is qualitatively the same as that of 3T3 cells, however, the transformed cells have a lower quantitative requirement. Both 3T3 and BP3T3 cells show a density-dependent response to EGF, but the shift in the dose response curve for BP3T3 cells at high cell density is smaller than that seen for 3T3 cells. One cause of the restricted growth of 3T3 cells at high cell density compared with BP3T3 cells is the increased concentration of growth factor needed for stimulation of 3T3 cells at higher cell densities. A lower rate of depletion of other growth factory by BP3T3 cells may also explain the smaller effect of cell density on the EGF response of these cells.     

Fibroblast growth regulatory factor inhibits DNA synthesis in BALB/c 3T3 cells by arresting in G1

A cell surface macromolecular component from quiescent BALB/c 3T3 mouse cells (designated fibroblast growth regulatory factor, FGRF) inhibits DNA synthesis and cell division in growing 3T3 cells. Addition of FGRF to synchronized populations of growing 3T3 cells in the late G1 or early S phase did not inhibit DNA synthesis in the immediate S phase. However, a significant inhibition was observed in the S phase of the next round of cell cycle. Cells exposed to the regulatory factor in late S/early G2 or early G1 showed reduced DNA synthesis in the upcoming S phase; the late S/early G2 cells were more sensitive to inhibition than the cells in the G1. Further, the regulatory factor delayed the progression of G0/G1-arrested cells into the next S phase. These results suggest that the physiological effect of FGRF is to arrest cells in early G1, thus preventing their entry into a new round of cell cycle. In contrast to untransformed 3T3 cells, mouse cells transformed by SV40 were not subjected to growth-arrest by the regulatory factor, although the transformed cells contain active FGRF that inhibits DNA synthesis in growing 3T3 cells.     

Mitogen response and cell cycle kinetics of Swiss 3T3 cells in defined medium: differences from human fibroblasts and effects of cell density

The mitogen requirement and proliferative response of Swiss 3T3 cells in serum-free, chemically defined culture medium were compared with those of early-passage human diploid fibroblasts. The effects of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, transferrin, and dexamethasone on cell-cycle parameters were measured using 5'-bromo-deoxyuridine-Hoechst flow cytometry. Swiss 3T3 cells differ from human fibroblasts in several ways: (1) Swiss 3T3 cells showed a much higher dependence on PDGF than human fibroblasts; the growth of the latter, but not of the former, could be stimulated by the combination of EGF, insulin, and dexamethasone to the full extent of that when PDGF was present; (2) in the absence of PDGF, insulin was an absolute requirement for Swiss 3T3 cells to initiate DNA synthesis, while a substantial proportion of human fibroblasts could enter DNA synthesis without exogenous insulin or IGF-I; and (3) in the absence of PDGF, increasing insulin concentration increased the cycling fraction of Swiss 3T3 cells without an appreciable effect on the rate of cell exit from G0/G1, while under similar culture conditions, insulin showed its major effect on regulation of the G1 exit rate of human fibroblasts, without much effect on the cycling fraction. In addition, the proliferative response of high-density versus low-density, arrested Swiss 3T3 cells showed that the interaction of mitogens varied with cell density. At high cell density, the PDGF requirement was consistent with the "competence/progression" cell-cycle model. This growth response was not seen, however, when cells were plated at low density.     

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

Abstract. 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 (G_D) at which the integrated control of proliferation and differentiation is mediated, the growth factor/serum deficiency arrest state (G_S), and the nutrient deficiency arrest state (G_N). Cells at states of reversible nonterminal differentiation (G_D?) 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 G_D, G_S and G_N 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 G_S, G_D, G_D?. 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 G_D? and TD states. Thus, the composition of proteins and phosphoproteins in mesenchymal stem cells serves to characterize different states of growth arrest and differentiation. The identification of differential protein expression provides an opportunity to test their functional role in growth and differentiation control.     

Multiplication of Swiss 3T3 cells in a serum-free medium

Gently trypsinized Swiss 3T3 cells inoculated into medium MCDB 402 attach readily to polylysine-coated surfaces and remain viable for several days in the absence of exogenously added protein. Short-term multiplication under defined conditions can be obtained by supplementing the MCDB 402 with fibroblast growth factor (FGF), insulin (INS), and dexamethasone (DEX). Addition of bovine plasma fibronectin further improves attachment and viability. This system does not require initial plating in serum or the addition of poorly defined extracts for cellular attachment or for multiplication. In the complete system minus FGF, cells plated at a low density attach to the culture surface and become quiescent. The addition of FGF or PDGF 48–72 h after plating stimulates a high level of DNA synthesis during the following 24 h. EGF also stimulates DNA synthesis in these cells, but to a lesser extent. Insulin and dexamethasone are not needed for the initial DNA synthesis response to FGF, but are needed for continuing multiplication over a period of several days. This system provides a means for studying the effects of specific mitogens on Swiss 3T3 cells in the absence of undefined supplements, and without complications due to density-dependent inhibition.     

Protein phosphorylation in a tetradecanoyl phorbol acetate-nonproliferative variant of 3T3 cells.

The 3T3-TNR9 cell line is a variant of Swiss 3T3 cells which does not respond mitogenically to tumor promoters, but does respond mitogenically to epidermal growth factor, fibroblast growth factor, and serum. To elucidate differences between tumor promoters and polypeptide mitogens in the pathway(s) of mitogenesis which might be responsible for the nonresponsiveness of the 3T3-TNR9 cells, we have examined in these cells the early protein phosphorylation events known to be associated with mitogenesis in the parental 3T3 cells. We find that the 3T3-TNR9 cells display levels of tetradecanoyl phorbol acetate binding and of a calcium- and phospholipid-dependent protein kinase activity which are at least the equal of those seen in the parental 3T3 cells, implicating some postreceptor event in the nonmitogenic phenotype. In addition, we find that phosphorylation of the epidermal growth factor receptor and of 80-kDa and 22-kDa proteins, as well as the tyrosine phosphorylation of a 42-kDa protein, all proceed normally in the nonmitogenic variant, even though these phosphorylations must depend on the activation of different kinases. Thus, all these early phosphorylation reactions are intact in the 3T3-TNR9 cells. Although these phosphorylations may be necessary, they clearly are insufficient to trigger mitogenesis.     

Induction of tumor promotor-inducible genes in murine 3T3 cell lines and tetradecanoyl phorbol acetate-nonproliferative 3T3 variants can occur through protein kinase C-dependent and -independent pathways.

We isolated a group of genes that are rapidly and transiently induced in 3T3 cells by tetradecanoyl phorbol acetate (TPA). These genes are called TIS genes (for TPA-inducible sequences). Epidermal growth factor (EGF), fibroblast growth factor (FGF), and TPA activated TIS gene expression with similar induction kinetics. TPA pretreatment to deplete protein kinase C activity did not abolish the subsequent induction of TIS gene expression by epidermal growth factor or fibroblast growth factor; both peptide mitogens can activate TIS genes through a protein kinase C-independent pathway(s). We also analyzed TIS gene expression in three TPA-nonproliferative variants (3T3-TNR2, 3T3-TNR9, and A31T6E12A). The results indicate that (i) modulation of a TPA-responsive sodium-potassium-chloride transport system is not necessary for TIS gene induction either by TPA or by other mitogens and (ii) TIS gene induction is not sufficient to guarantee a proliferative response to mitogenic stimulation.     

Role of cell density/cell-cell contact,and growth state in expression of differentiated properties by the LLC-PK1 Cell

Populations of the renal epithelial cell line, LLC-PK,₁, acquire many properties characteristic of the proximal tubular cell at confluence. At confluence cells both enter a nonproliferative state and develop extensive cell-cell contacts. To determine if one or both factors is responsible for acquisition of the differentiated phenotype, growth arrest was initiated in populations of varying densities by two procedures (serum deprivation and thymidine block) and expression of several differentiated properties (Na-hexose symport activity, gamma-glutamyl transpeptidase activity, alkaline phosphatase activity, and villin protein) was examined. Induction of growth arrest resulted in expression of all differentiated properties even in subconfluent populations. The level of expression in a population was proportional to cell density at the initiation of growth arrest; higher density was associated with increased expression. Evidence indicated the existence of some minimal density below which cells could not express detectable levels of differentiated properties in response to induction of growth arrest. The procedure used to initiate growth arrest did not affect this behavior, indicating that initiation of cell growth arrest rather than hormone deprivation was the inducing factor. These results indicate that both cell growth state and cell density independently modulate expression of differentiated properties by the LLC-PK₁ cell. These results are incorporated into a model in which cells in the absence of “appropriate” cell-cell contact arrest at a differentiation-incompetent cell cycle point. In the presence of appropriate cell-cell contact (as yet undefined) cells arrest at a distinct differentiation-competent cell cycle point and initiate expression of the differentiated phenotype. © 1994 wiley-Liss, Inc.     

神经节苷脂GD3与肿瘤的血管生成作用(英文)

 血管生成作用 (angiogenesis)是实体瘤 (solidtumor)生长和扩散的必要条件 .实体瘤的微血管密度与肿瘤的恶性程度成正相关 ,而且也与病人的预后密切相关 .因此 ,对抗血管生成作用是一种很有吸引力的肿瘤疗法 .神经节苷脂GD3在多种类型的肿瘤中超常表达 .一般认为 ,神经节苷脂GD3有增强肿瘤本身及邻近组织中的血管生成作用 ,从而促进肿瘤的演进和转移 .最近的研究工作为这一假设提供了有力的实验证据 .应用GD3合酶的反意DNA转染肿瘤细胞从而抑制细胞中的GD3合酶的表达 ,极大地降低了细胞的内源GD3含量 .进一步的研究证明 ,抑制肿瘤细胞的GD3合成明显地降低了该肿瘤细胞的血管内皮生长因子 (VEGF)的水平 ,并使血管生成作用降至最小限度 .这些实验说明GD3在肿瘤的血管生成中具有重要的作用 .此外 ,GD3作为肿瘤的一种相关抗原 ,它与血管生成因子的协同效应将在未来的联合基因疗法中起到重要的作用     

3T3-L1 preadipocyte differentiation and poly(ADP-ribose) synthetase

Differentiation of 3T3-L1 preadipocytes, induced by methyl-isobutylxanthine (MIX), dexamethasone (DEX), and insulin, results in cells with the morphological and biochemical characteristics of adipocytes. Following incubation of 3T3-L1 cells with MIX, DEX, and insulin, poly(ADP-ribose) synthetase activity decreased abruptly, remained low for several hours and then increased; this rise was delayed by readdition of MIX, DEX, and insulin. The transient reduction in poly(ADP-ribose) synthetase activity in 3T3-L1 cells occurred prior to the appearance of the adipocyte phenotype induced by the above agents. It was not observed when preparations were assayed in the presence of DNase I, indicating that poly(ADP-ribose) synthetase activity was masked following treatment with MIX, DEX, and insulin. The change in synthetase activity represents the earliest alteration of a specific enzyme yet detected during the differentiation of 3T3-L1 cells. It appears to be differentiation specific since nondifferentiating 3T3-C2 control cells did not exhibit changes in poly(ADP-ribose) synthetase activity when treated with MIX, DEX, and insulin. The transient reduction in activity may be an early event in differentiation which reflects changes in chromatin structure.     

Antidiabetic AD4743 enhances adipocyte differentiation of 3T3 T mesenchymal stem cells

AD4743 is an antidiabetic agent that, when added to fetal bovine serum (FBS), has been shown to have adipogenic activity for some proadipocyte cell lines once they reach confluence. In the current study, the effects of AD4743 on the growth and adipocytic differentiation of 3T3 T multipotential mesenchymal stem cells have been tested. 3T3 T cells, unlike other cells capable of undergoing adipocyte differentiation, are routinely induced to differentiate at low cell density. This is done using platelet-poor human plasma (HP), a potent inducer of growth arrest and differentiation. AD4743 (0-200 micrograms/ml) was tested in varied concentrations of HP or FBS, at varied cell densities, and at various times during growth and differentiation. AD4743 slowed the growth rate of 3T3 T cells and it induced their differentiation in a dose-dependent manner in medium containing 10% FBS once they reached confluence. The data suggest that the ability of AD4743 to inhibit growth may also be coupled with its ability to enhance differentiation. In addition, AD4743 (1-10 micrograms/ml) in the presence of 25% HP markedly increased the kinetics of adipocyte differentiation, at low (less than 5,000 cells/cm2) or high cell density. Greater than 50% cell differentiation could be achieved in 2 days in low density cultures; 80-95% differentiation could be achieved in just 4 days, compared to 8-12 days in a typical culture. The maximum amount of differentiation in HP was potentiated by AD4743 to a greater degree in poor lots of HP; however, the kinetics were increased in all lots. Adipocytic differentiation was measured both morphologically and by Northern blot analyses of differentiation-specific genes. AD4743 at 1-10 micrograms/ml appeared to be most effective, depending on the cell density and other conditions. The mechanism of action of AD4743 remains to be elucidated, but the enhancement of adipocyte differentiation does not appear to occur via an insulin-dependent pathway.