Cell proliferation is accompanied by an increase in the utilization of glucose and glutamine. The proliferative response is dependent on a decrease in the activity of the ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C)-Cdh1 which controls G1-to-S-phase transition by targeting degradation motifs, notably the KEN box. This occurs not only in cell cycle proteins but also in the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), as we have recently demonstrated in cells in culture. We now show that APC/C-Cdh1 controls the proliferative response of human T lymphocytes. Moreover, we have found that glutaminase 1 is a substrate for this ubiquitin ligase and appears at the same time as PFKFB3 in proliferating T lymphocytes. Glutaminase 1 is the first enzyme in glutaminolysis, which converts glutamine to lactate, yielding intermediates for cell proliferation. Thus APC/C-Cdh1 is responsible for the provision not only of glucose but also of glutamine and, as such, accounts for the critical step that links the cell cycle with the metabolic substrates essential for its progression.cell cycle | glutaminase | 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 | proliferation H uman blood T lymphocytes have been used for many years in studies of cell proliferation (1, 2). These cells can be obtained directly from the circulation and therefore avoid the pitfalls associated with the use of cells in culture, which acquire confounding characteristics as a result of their in vitro environment (3). Interest has recently been rekindled in the metabolic changes that underpin cell proliferation in cancer to identify potential targets for chemotherapy (4,5). This has highlighted the need to carry out comparative studies on proliferating normal and tumor cells to ascertain whether an antimetabolic approach to cancer is possible without side effects related to the mechanism of action.The E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) attached to the activator protein Cdh1 plays a crucial role in controlling G1-to S-phase transition, and therefore proliferation, through the breakdown of cell cycle proteins (6, 7). APC/C-Cdh1 substrates are targeted for degradation through specific recognition motifs, including one known as the KEN box (8). Inactivation of APC/C-Cdh1 in G1 of the cell cycle is necessary for initiation of S phase, in which DNA is replicated and chromosomes are duplicated. We have recently found that APC/CCdh1 links cell cycle activity with that of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) (9, 10). PFKFB3-a key regulator of glycolysis (11)-contains a KEN box motif and is thus also broken down by APC/C-Cdh1. Inactivation of APC/C-Cdh1 enables PFKFB3 to up-regulate glycolysis, thus providing the cell with the glucose essential for the subsequent biosynthesis of macromolecules. Our previous studies (9) were carried out in two cell lines; we therefore decided to investigate whether the same mechanis...

Gangliosides have been involved in multiple cellular processes such as growth, differentiation and adhesion, and more recently as regulators of cell death signaling pathways. Some of these molecules can be considered as tumor-associated antigens, in particular, N-glycolyl sialic acid -containing gangliosides, which are promising candidates for cancer-targeted therapy because of their low expression in normal human tissues. In this study, we provided the molecular and cellular characterization of a novel cell death mechanism induced by the anti-NGcGM3 14F7 monoclonal antibody (mAb) in L1210 murine tumor cell line but not in mouse normal cells (B and CD4 + T lymphocytes) that expressed the antigen. Impairment of ganglioside synthesis in tumor cells abrogated the 14F7 mAb cytotoxic effect; however, exogenous reincorporation of the ganglioside did not restore tumor cell sensitivity to 14F7 mAb-induced cytotoxicity. 14F7 F(ab ¶) 2 but not Fab fragments retained the cytotoxic capacity of the whole mAb. By contrary, other mAb, which recognizes N-glycolylated gangliosides, did not show any cytotoxic effect. These mAbs showed quite different capacities to bind NGcGM3-positive cell lines measured by binding inhibition experiments. Interestingly, this complementindependent cell death mechanism did not resemble apoptosis, because no DNA fragmentation, caspase activation, or Fas mediation were observed. However, NGcGM3 ganglioside-mediated 14F7 mAb-induced cell death was accompanied by cellular swelling, membrane lesion formation, and cytoskeleton activation, suggesting an oncosislike phenomenon. This novel mechanism of cell death lets us to support further therapeutic approaches using NGcGM3 as a molecular target for antibody-based cancer immunotherapy.

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Scavenger receptor cysteine-rich (SRCR) domains are evolutionally conserved modules that display complex structures stabilized by key amino acids, while some other residues have evolved with a relative independence, thus allowing the functional diversity of these receptors. CD6, a highly glycosylated membrane protein predominantly expressed on lymphocytes, contains three SRCR domains. The lack of CD6 domain crystal structure has limited the characterization of the binding sites for the interacting molecules. The interaction between CD6 and its ligand, activated leukocyte-cell adhesion molecule (ALCAM)/CD166, through the membrane-proximal SRCR3 domain, has low affinity and involves conserved sites in both molecules mediating a cross-species binding. The CD6-ALCAM interaction has been involved in cell adhesion, maturation, regulation of activation, and survival processes, suggesting the potential relevance of this target for therapeutic interventions. Several anti-CD6 monoclonal antibodies (MAb) have been described but their affinity and epitope definition remain unclear. We found the murine and humanized T1 MAb versions have similar CD6 recognition profiles and affinity constants of about 6 x 10(8). These antibodies do not block the CD6-ALCAM interaction and recognize a conformational epitope independent of the CD6 N-glycosylation. This epitope was additionally found in the chimpanzee and contains an RXE/Q consensus motif located in the membrane-distal SRCR1. These results, together with the therapeutic evidence previously obtained with these MAbs, suggest a differential contribution of CD6 domains to lymphocyte biology. Potential mechanisms for T1 MAb therapeutic effect different from CD6-CD166 interaction blocking would be dissected.

While 'proof of concept' Phase II and III clinical trials with the NGcGM3/VSSP vaccine in cancer patients are currently ongoing these results reasonably sustain the validation of this peculiar ganglioside as a novel target for cancer immunotherapy.