Autophagic cell death (ACD) can be operationally described as cell death with an autophagic component. While most molecular bases of this autophagic component are known, in ACD the mechanism of cell death proper is not well defined, in particular because in animal cells there is poor experimental distinction between what triggers autophagy and what triggers ACD. Perhaps as a consequence, it is often thought that in animal cells a little autophagy is protective while a lot is destructive and leads to ACD, thus that the shift from autophagy to ACD is quantitative. The aim of this article is to review current knowledge on ACD in Dictyostelium, a very favorable model, with emphasis on (1) the qualitative, not quantitative nature of the shift from autophagy to ACD, in contrast to the above, and (2) random or targeted mutations of in particular the following genes: iplA (IP3R), TalB (talinB), DcsA (cellulose synthase), GbfA, ugpB, glcS (glycogen synthase) and atg1. These mutations allowed the genetic dissection of ACD features, dissociating in particular vacuolisation from cell death.

Transforming growth factor-beta (TGFb)1 is thought to be implicated in breast cancer progression. However, data about the influence of TGFb1 on breast cancer development are conflicting. To clarify the clinical relevance of TGFb1, TGFb1 protein level has been measured by enzyme-immoassay in 193 breast tumour samples. We found that 94.3% of patients expressed TGFb1 with a range of 0 -684 pg mg À1 protein.In the overall population, an increase of tumoral TGFb1 was observed in premenopausal patients when compared to postmenopausal subgroup (P ¼ 0.0006). When patients were subdivided according to nodal status, TGFb1 was correlated to type-1 plasminogen activator inhibitor in the node-negative subgroup (P ¼ 0.040). Multivariate analysis revealed that, after lymph node status (P ¼ 0.0002) and urokinase-type plasminogen activator (P ¼ 0.004), TGFb1 was an independent prognostic marker for DFS (P ¼ 0.005) in the overall population. In the node-negative population, TGFb1 was the prominent prognostic factor (P ¼ 0.010). In the same population, Kaplan -Meier curves demonstrated that high TGFb1 level was correlated with a shorter diseasefree survival (P ¼ 0.020). These data suggest that the measurement of tumoral TGFb1 protein level, especially for node-negative patients, might help to identify a high-risk population early in tumour progression.

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Summary Amphiregulin (AR) is a heparin-binding epidermal growth factor (EGF)-related peptide that seems to play an important role in mammary epithelial cell growth regulation. We have investigated the regulation of AR-gene expression and -protein secretion by EGF in normal breast epithelial cells (HMECs), as well as in the tumoral breast epithelial cell lines MCF-7 and MDA-MB231. EGF induced a dosedependent increase of AR mRNA level in both normal and tumoral cells. Thus, 10 Ϫ8 M EGF stimulated AR expression in HMECs to 140-300% of control. A similar EGF concentration increased AR mRNA level to 550% and 980% of control in MCF-7 and MDA-MB231 cells, respectively. This was accompanied by an accumulation of AR into conditioned culture media. However, HMECs secreted in response to EGF, 5-10 fold more AR than tumour cells. Furthermore, the potential participation of AR in the regulation of the plasminogen activator (PA)/plasmin system was investigated. Whereas HMEC-proliferation was stimulated by AR, the levels of secreted urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAi-1) remained unaffected. Conversely, AR failed to regulate the proliferation of tumoral cell lines but induced an accumulation of uPA and PAi-1 into culture media. This was accompanied by an increase of the number of tumoral cells that invaded matrigel in vitro. Moreover, the presence of a neutralizing anti-uPA receptor antibody reversed the increased invasiveness of MDA-MB231 cells induced by AR. These data reveal differential behaviour of normal versus tumoral breast epithelial cells in regard to the action of AR and demonstrate that, in a number of cases, AR might play a significant role in tumour progression through the regulation of the PA/plasmin system. Whereas the expression of AR in a variety of both nontransformed and tumoral cells appears to be stimulated in the presence of EGF (Normanno et al, 1994b;Sehgal et al, 1994), the potential regulation of AR by growth factors in normal breast epithelial cells has never been examined. Because previous studies suggest that dysregulated expression of AR may be a component of mammary tumorigenesis we proposed to analyse and to compare the regulation of amphiregulin gene expression and protein secretion by EGF in normal and tumoral breast epithelial cells. Moreover, our aim was to examine potential role of AR in the regulation of uPA and PAi-1 protein secretion that could account for breast cancer progression. MATERIAL AND METHODS MaterialsAnti-smooth muscle α-actin, -actin, -vimentin and -cytokeratin 14, 18 and 19 antibodies were provided by Sigma (St Quentin Fallavier, France); Anti-vimentin antibody was from DAKO (Denmark). The AR cDNA probe was obtained from Dr. Plowman (Bristol-Myers Squibb, Seattle, WA). Matrigel was provided by Becton-Dickinson (France). Anti-uPA receptor neutralizing antibody, recombinant human AR and AR enzyme-immunoassay kit were from R & D Systems (Abingdon, UK). Antibodies used in ELISA determination of AR recognize various forms of...

Two inducers, DIF-1 and c-di-GMP, each separately shown to play a major role in Dictyostelium cell death induction in vitro, in fact cooperate. A similar cooperation with polyketides might occur for c-di-GMP effects in other situations and organisms, in particular in innate immunity and cell death in animal cells.

Autophagic cell death in Dictyostelium can be dissociated into a starvation-induced sensitization stage and a death induction stage. A UDP-glucose pyrophosphorylase (ugpB) mutant and a glycogen synthase (glcS) mutant shared the same abnormal phenotype. In vitro, upon starvation alone mutant cells showed altered contorted morphology, indicating that the mutations affected the pre-death sensitization stage. Upon induction of cell death, most of these mutant cells underwent death without vacuolization, distinct from either autophagic or necrotic cell death. Autophagy itself was not grossly altered as shown by conventional and electron microscopy. Exogenous glycogen or maltose could complement both ugpB(-) and glcS(-) mutations, leading back to autophagic cell death. The glcS(-) mutation could also be complemented by 2-deoxyglucose that cannot undergo glycolysis. In agreement with the in vitro data, upon development glcS(-) stalk cells died but most were not vacuolated. We conclude that a UDP-glucose derivative (such as glycogen or maltose) plays an essential energy-independent role in autophagic cell death.

Through random mutagenesis, the receptor histidine kinase DhkM was found essential for autophagic cell death (ACD) in Dictyostelium. DhkM is the most downstream known molecule required for this model ACD. Different DhkM mutants showed distinct non-vacuolizing ACD phenotypes and genetically separated previously undissociated late cell death events.