Milk fat globule--epidermal growth factor--factor VIII (MFGE8)/lactadherin promotes bladder tumor development.

Milk fat globule-epidermal growth factor-factor VIII (MFGE8), also called lactadherin or SED1, is a secreted integrin-binding protein that promotes elimination of apoptotic cells by phagocytes leading to tolerogenic immune responses, and vascular endothelial growth factor (VEGF)-induced angiogenesis: two important processes for cancer development. Here, by transcriptomic analysis of 228 biopsies of bladder carcinomas, we observed overexpression of MFGE8 during tumor development, correlated with expression of genes involved in cell adhesion or migration and in immune responses, but not in VEGF-mediated angiogenesis. To test whether MFGE8 expression was instrumental in bladder tumor development, or a simple consequence of this development, we used genetic ablation in a mouse model of carcinogen-induced bladder carcinoma. We showed that Mfge8 was also upregulated in mouse carcinoma, and that in its absence, Mfge8-deficient animals developed less advanced tumors. Angiogenesis was similar in carcinogen-treated Mfge8-expressing or -deficient bladders, thus ruling out a major role of the proangiogenic function of Mfge8 for its protumoral role. By contrast, the tumor-promoting role of Mfge8 was not observed anymore in mice devoid of adaptive immune system, and human tumors overexpressing MFGE8 where invaded with macrophages and regulatory T cells, thus suggesting that MFGE8/lactadherin favors development of bladder tumors at least partly by an immune system-dependent mechanism. Our observations suggest future use of MFGE8-inhibiting molecules as therapies of bladder carcinomas, and of a limited number of other human cancers, in which our analysis of public databases also revealed overexpression of MFGE8.

Inhibition of angiotensinogen production by angiotensin II analogues in human hepatoma cell line.

The aim of this study was to examine in Hep G2, a human hepatoma-derived cell line, the presence of angiotensin II (ANG II) receptors and the effect of ANG II and its analogues on angiotensinogen production. The presence of ANG II receptors was demonstrated using a long-acting ANG II analogue, 125I-labeled [Sar1]ANG II. A single class of specific binding sites was identified in these cells with a dissociation constant (Kd) of 2 nM. The number and affinity of these binding sites were not changed by [Sar1]ANG II treatment over 24 h. ANG II showed an inhibitory effect on angiotensinogen production. [Sar1]ANG II also exhibited a similar inhibitory effect as that of ANG II but to a greater extent and therefore was used throughout these studies. [Sar1]ANG II inhibited angiotensinogen production in a dose-dependent manner, exhibiting a half-maximal inhibitory concentration (IC50) of 2 nM. Other ANG II analogues showed similar effects on angiotensinogen production. In order of decreasing ability, they were [Sar1]ANG II greater than [Sar1-Ala8]ANG II greater than [Sar1-Val8]ANG II greater than [Sar1-Val5-(Br5)-Phe8]ANG II greater than [Sar1-Val5-DPhe8]ANG II. Results of these studies show that the Hep G2 cell possesses specific ANG II receptors and that [Sar1]ANG II induces a dose-dependent inhibition of angiotensinogen production in this system.

Regulation of angiotensinogen production by angiotensin II analogues.

Specific binding sites for angiotensin II (Ang II) were identified in a human hepatoma cell line, HepG2. Binding of [125I]-Sar1 Ang II to these cells showed a high-affinity site with a Kd of 2.4 +/- 0.2 nmol/l. This specific binding was not changed during the cell cycle and showed no alteration after 24 h of treatment with Sar1-Ang II (10(-8) mol/l). Exposure of HepG2 cells to the Ang II agonist Sar1-Ang II caused a dose-dependent decrease in angiotensinogen production. The maximal inhibitory effect was at a dose of 10(-6) mol/l Sar1-Ang II which elicited 67% inhibition of angiotensinogen production after 24 h (control: 2.015 +/- 0.5 micrograms angiotensinogen/mg DNA; Sar1-Ang II 10(-6) mol/l: 0.68 +/- 0.03 micrograms angiotensinogen/mg DNA). Fifty per cent inhibition was obtained at a dose of 10(-9) mol/l Sar1-Ang II. Angiotensin II had a less marked effect, showing maximal inhibition of 40%. This study shows that the HepG2 cells possess specific Ang II binding sites and that Ang II analogues induce a dose-dependent inhibition of angiotensinogen production in cell culture.

Induction of human T colony formation by phorbol myristate acetate.

Phorbol myristate acetate (PMA) is a potent inducer of T colony formation by peripheral blood lymphocytes. A mean cloning efficiency of 0.3% (0.05-0.5%) is obtained with PMA concentrations of 100-1000 ng/ml. PMA-induced T colony formation does not require the presence of monocytes and therefore differs from other mitogens in this respect. Purified T-colony-promoting activity (TCPA) (devoid of phytohaemagglutinin (PHA)) increases PMA-induced T colony numbers and induces T colony formation at low PMA doses (0.01 to 1 ng), concentrations at which no T colonies are detected in the absence of added TCPA. PMA-induced colonies are mainly composed of cells bearing Fc receptors for IgM (54%), which is not the case for colonies obtained with PHA (11%). PMA-induced colony cells do not bind OKT3 and OKT4 monoclonal antibodies, whereas 23% are able to bind OKT8 antibody. These results demonstrate that PMA is a potent inducer of T colony formation and may therefore serve as a useful tool for the study of T-cell differentiation.

Modulation of PHA-induced T colony formation by phorbol myristic acetate.

PMA (10 ng/ml), induced a four-fold increase in PHA-induced T colony formation by peripheral blood lymphocytes (PBL). At lower (0.1-1 ng/ml) and higher concentrations (100-1,000 ng/ml), PMA had an inhibitory effect. The potent co-mitogen effect observed at 10 ng/ml PMA was associated with a strong increase in T colony promoting activity (TCPA) released by PHA stimulated PBL cultured in the presence of PMA (10 ng/ml). PMA at all concentrations exerted an inhibitory effect on T colony formation when PBL were cultured in the presence of PHA and an optimal concentration of exogenous TCPA. PMA was also capable of assuming the essential role played by monocytes in T colony formation. Thus, PMA can fulfil both co-mitogen and monocyte like roles in T colony formation. These activities closely resemble those previously described for interleukin 2 (IL2) production.