Epidermal growth factor (EGF) at 3 nM maximally inhibits the proliferation of A431 epidermoid carcinoma cells. We show that at lower concentrations, in the range of 3-100 pM, EGF has a mitogenic effect on A431 cells. In Epidermal growth factor (EGF) promotes the growth of many cell types in vitro (1-3) and inhibits proliferation of several cell types-e.g., GH4 rat pituitary tumor cells (4), A431 epidermoid carcinoma cells (5, 6), and certain human breast cancer cells (7). EGF initially binds to receptors homogeneously distributed on the cell surface. Subsequent events have been described by various investigators and include receptor phosphorylation, aggregation, internalization, and degradation in lysosomes (1). The mechanism by which these events induce DNA synthesis and cytokinesis is unknown.It has been found that at least 6-8 hr of EGF exposure are required to stimulate DNA synthesis (8). Das and Fox have suggested that EGF-induced internalization and degradation of the EGF receptor are rate-limiting factors for EGF-induced mitogenesis (9, 10), perhaps through production of a second messenger. Recent studies showed enhancement of EGF stimulation of DNA synthesis by amine compounds, which inhibited clustering of receptors in coated pits (11), and by phorbol esters, which reduced both the affinity of EGF receptors for EGF and its subsequent degradation (12)(13)(14). These results suggest that EGF stimulation of cell growth might only require the presence of EGF-EGF receptor complexes at the cell surface in contradiction to the above hypothesis.Shechter et al., on the other hand, suggested that the stimulatory effect of EGF might be mediated by small amounts of high-affinity EGF receptors, which remain at the cell surface for more than 8 hr when occupied by EGF (15). King and Cuatrecasas also have suggested that the accumulation of stable intracellular complexes between high-affinity receptors and EGF are involved in growth stimulation, but the role of these highaffinity receptors in mitogenesis remains unclear (16).A431 cells lend themselves to the study of EGF interactions with receptors because of their extremely high number of EGF receptors (1-3 x 10' per cell) (1,17,18

A major limitation in developing applications for the use of human embryonic stem cells (HESCs) is our lack of knowledge of their responses to specific cues that control self-renewal, differentiation, and lineage selection. HESCs are most commonly maintained on inactivated mouse embryonic fibroblast feeders in medium supplemented with FCS, or proprietary replacements such as knockout serum-replacement together with FGF-2. These undefined culture conditions hamper analysis of the mechanisms that control HESC behavior. We have now developed a defined serum-free medium, hESF9, for the culture of HESCs on a type I-collagen substrate without feeders. In contrast to other reported media for the culture of HESCs, this medium has a lower osmolarity (292 mosmol/liter), l -ascorbic acid-2-phosphate (0.1 μg/ml), and heparin. Insulin, transferrin, albumin conjugated with oleic acid, and FGF-2 (10 ng/ml) were the only protein components. Further, we found that HESCs would proliferate in the absence of exogenous FGF-2 if heparin was also present. However, their growth was enhanced by the addition of FGF-2 up to 10 ng/ml although higher concentrations were deleterious in the presence of heparin.

Monoclonal antibodies penetrate bulky tumors poorly after intravenous administration, in part because of specific binding to the target antigen. Experiments presented here demonstrate an analogous phenomenon in micrometastases; poor antibody penetration, attributable to a "binding-site barrier" phenomenon, can be seen in guinea pig micrometastases as small as 300 ,um in diameter. Increasing the dose of antibody can partially overcome this limitation, but at a cost in specificity. Six and 72 hr after injection, animals were sacrificed by CO2 inhalation. Organs were rapidly removed and weighed, and tissue radioactivity was determined. Lung tissues with metastases were frozen immediately in OCT compound (Tissue-Tek, Miles) and then processed for autoradiography and immunostaining. For high-dose experiments, 1000 ,ug of unlabeled D3 was added to 30 ,ug of labeled D3. Autoradiography and Immunostaining. Serial 20-,um sections were cut from the frozen lung tissues. For autoradiography, sections were placed on SB-5 film (Eastman Kodak) and exposed for 1-4 days. When 125I-labeled BL3 was coinjected with I311-labeled D3, images of the latter were obtained shortly after sacrifice, images of the former after eight 131I half-lives.The avidin-biotin peroxidase complex (ABC) method (29) was used to compare Ab and Ag distribution. Briefly, tissue sections were acetone-fixed for 10 min and incubated with blocking serum for 20 min. To assess Ag expression, sections were incubated with D3 Ab (20 ,ug/ml) for 1 hr at room temperature, biotinylated horse anti-mouse IgG for 45 min, ABC for three 30-min periods, and diaminobenzidine (Vector Laboratories). When injected D3 was assessed, incubation with D3 was omitted. Some sections were also stained for blood vessels by use of polyclonal rabbit anti-factor VIII and an alkaline phosphatase substrate, Vector Red (30). RESULTS AND DISCUSSIONIn this study, we have assessed the distribution of i.v. administered mAbs in experimental guinea pig lung micrometastases. Two weeks after i.v. injection of L10 cells, multiple metastases 100-1000 ,um in diameter could be seen in the lungs of all animals. This model (metastasis of guinea pig cancer in guinea pigs) is in many ways more relevant to the Abbreviations: Ab, antibody; mAb, monoclonal Ab; Ag, antigen.

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We have developed a serum-free medium, designated ESF7, in which leukemia inhibitory factor (LIF) clearly stimulated murine embryonic stem (ES) cell proliferation accompanied by increased expression of nanog and Rex-1 and decreased FGF-5 expression. These effects were dependent on the concentration of LIF. The ES cells maintained in ESF7 medium for more than 2 yr retained an undifferentiated phenotype, as manifested by the expression of the transcription factor Oct-3/4, the stem cell marker SSEA-1, and alkaline phosphatase. Withdrawal of LIF from ESF7 medium resulted in ES cell apoptosis. Addition of serum to ESF7 medium promoted ES cell differentiation. Addition of BMP4 promoted ES cell differentiation into simple epithelial-like cells. In contrast, FGF-2 promoted ES cell differentiation into neuronal and glial-like cells. Under serum-free culture conditions, LIF was sufficient to stimulate cell proliferation, it inhibited cell differentiation, and it maintained self-renewal of ES cells. Because this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent ES cells in vitro, it will allow the elucidation of ES cell responses to growth factors under defined conditions.

1 We examined regeneration of endothelial cells (ECs), neointima formation, decreased endotheliumdependent relaxation (EDR) and changes in the contents of L-arginine, NG-monomethyl-L-arginine (L-NMMA), asymmetrical NG, N0-dimethylarginine (ADMA) and symmetrical NG,NG-dimethylarginine (SDMA) in the regnerated ECs, 6 weeks after balloon denudation of the rabbit carotid artery. 2 Regeneration of ECs was completed in 6 weeks and a significant neointima formation accompanied by the decreased EDR was observed. 3 L-NMMA and ADMA contents in the regenerated ECs (23.5 + 4.3 and 21.2 + 2.0 pmol mg-' DNA, respectively) were significantly (P <0.05 and P <0.01) higher than those in the control ECs (8.8 + 3.0 and 7.4 + 1.9 pmol mg'l DNA, respectively), whereas L-arginine was significantly (P < 0.005) decreased in the regenerated ECs (31,470+1,050 pmol mg-' DNA) as compared to that in the control ECs (47,870+ 1,890 pmol mg-' DNA). SDMA content was below the assay limits. 4 L-NMMA and ADMA, but not SDMA, inhibited the EDR induced by acetylcholine in a concentration-dependent manner. The inhibition with L-NMMA and ADMA was prevented by an addition of L-arginine, but not by D-arginine. 5 These results suggest that the accumulation of endogenous inhibitors for nitric oxide synthesis and decreased L-arginine content are associated with decreased NO production/release from regenerated ECs and neointima formation. Keywords: Neointima formation; endogenous inhibitors for NO synthesis; balloon denudation; decreased endotheliumdependent relaxation; EDRF/NO Introduction MethodsAs proposed by Ross & Glomset (1976a,b) and Ross (1986), denudation and nondenudation intimal injuries may play an important role in the development of atherosclerotic vascular disease (Ip et al., 1990). We have reported that restoration of the endothelium after balloon denudation of the rabbit carotid artery was completed in 6 weeks and that regenerated ECs were less capable of producing/releasing the endothelium-derived relaxing factor (EDRF/nitric oxide: NO) (Azuma et al., 1990;Niimi et al., 1994). EDRF/NO inhibits platelet adhesion and aggregation (Azuma et al., 1986;Radomski et al., 1987), which is considered as an initiating event for neointima formation (Ross, 1986;Schwartz et al., 1981). It has also been demonstrated that NO inhibits smooth muscle cell proliferation (Garg & Hassid, 1989). These findings when considered together suggest that the neointima formation after endothelial removal might be caused at least partly through impairment of the ability of ECs to produce/release EDRF/NO. However, the mechanism by which the production/release of EDRF/NO is reduced remains unclear. Recently, Vallance et al. (1992) have obtained evidence NG-monomethyl-L-arginine (L-NMMA) and asymmetrical NG,NG-dimethyl arginine (ADMA) play a role as endogenous inhibitors of NO synthesis. The present experiments were, therefore, designed to determine the regeneration of ECs, the neointima formation, the decreased EDR and the changes in content of L-arginine, L-NMMA, ADMA an...

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A distinct family of endothelial cell mitogens that are homologous to platelet-derived growth factor has recently been identified. Unlike other known endothelial cell mitogens, these vascular endothelial cell growth factors (VEGFs) are secreted and appear to act specifically on endothelial cells. We have purified VEGF 2083-fold to apparent homogeneity from protein-free culture medium conditioned by