Since HLA‐E heavy chains accumulate free of their light β₂‐microglobulin (β₂m) subunit, raising mAbs to folded HLA‐E heterodimers has been difficult, and mAb characterization has been controversial. Herein, mAb W6/32 and 5 HLA‐E‐restricted mAbs (MEM‐E/02, MEM‐E/07, MEM‐E/08, DT9, and 3D12) were tested on denatured, acid‐treated, and natively folded (both β₂m‐associated and β₂m‐free) HLA‐E molecules. Four distinct conformations were detected, including unusual, partially folded (and yet β₂m‐free) heavy chains reactive with mAb DT9. In contrast with previous studies, epitope mapping and substitution scan on thousands of overlapping peptides printed on microchips revealed that mAbs MEM‐E/02, MEM‐E/07, and MEM‐E/08 bind three distinct α1 and α2 domain epitopes. All three epitopes are linear since they span just 4–6 residues and are “hidden” in folded HLA‐E heterodimers. They contain at least one HLA‐E‐specific residue that cannot be replaced by single substitutions with polymorphic HLA‐A, HLA‐B, HLA‐C, HLA‐F, and HLA‐G residues. Finally, also the MEM‐E/02 and 3D12 epitopes are spatially distinct. In summary, HLA‐E‐specific residues are dominantly immunogenic, but only when heavy chains are locally unfolded. Consequently, the available mAbs fail to selectively bind conformed HLA‐E heterodimers, and HLA‐E expression may have been inaccurately assessed in some previous oncology, reproductive immunology, virology, and transplantation studies.

Mitotane inhibits steroid synthesis by an action on steroidogenic enzymes, as 11β-hydroxylase and cholesterol side chain cleavage. It also has a cytotoxic effect on the adrenocortical cells and represents a primary drug used in the adrenocortical carcinoma (ACC). H295R and SW13 cell lines were treated with mitotane 10-5 M and ionizing radiations (IR) in combination therapy, inducing an irreversible inhibition of cell growth in both adrenocortical cancer cells. As shown in a previous report, mitotane/IR combination treatment induced a cell accumulation in the G2 phase. Here, we report the radiosensitizing properties of mitotane in two different ACC cell lines. The drug reveals the effectiveness to enhance the cytotoxic effects of IR by attenuating DNA repair and interfering on the activation of mitosis promoting factor (MPF), mainly regulated by the degradation of cyclin B1 in the mitotic process. These events may explain the inappropriate activation of cdc2, implicated in G2/M phase arrest and probably induced by the mitotane and IR in the combined treatment. Indeed, treatment with purvalanol, a cdc2-inhibitor prevents cell cycle arrest, triggering the G2/M transition. The observation that mitotane and IR in combination treatment amplifies the activation level of cyclin B/cdc2 complexes contributing to cell cycle arrest, suggests that the MPF could function as a master signal for controlling the temporal order of different mitotic events. Moreover, we report that mitotane interferes in modulation of mismatch repair (MMR) enzymes, revealing radiosensitizing drug ability

We report on the use of one-dimensional photonic crystals to detect clinically relevant concentrations of the cancer biomarker ERBB2 in cell lysates. Overexpression of the ERBB2 protein is associated with aggressive breast cancer subtypes. To detect soluble ERBB2, we developed an optical set-up which operates in both label-free and fluorescence modes. The detection approach makes use of a sandwich assay, in which the one-dimensional photonic crystals sustaining Bloch surface waves are modified with monoclonal antibodies, in order to guarantee high specificity during the biological recognition. We present the results of exemplary protein G based label-free assays in complex biological matrices, reaching an estimated limit of detection of 0.5 ng/mL. On-chip and chip-to-chip variability of the results is addressed too, providing repeatability rates. Moreover, results on fluorescence operation demonstrate the capability to perform high sensitive cancer biomarker assays reaching a resolution of 0.6 ng/mL, without protein G assistance. The resolution obtained in both modes meets international guidelines and recommendations (15 ng/mL) for ERBB2 quantification assays, providing an alternative tool to phenotype and diagnose molecular cancer subtypes.

C-MYC is overexpressed in many types of cancer linked to poor prognosis. We examined the c-Myc protein expression in adrenocortical cancer (ACC) cells to investigate the role of this protein in the neoplasm, its involvement in chemotherapy and finally to determine whether c-Myc could be considered a prognostic factor in patients with ACC. H295R and SW13 cell lines were treated with paclitaxel. c-Myc overexpressing cell clones were achieved by transfecting the H295R cell line with the pcDNA3-hMYC plasmid expressing the full-lengh C-MYC coding sequence. The SW13 cell line was transfected with siRNA oligonucleotides for C-MYC. Cell cycle analysis was evaluated by flow cytometry. c-Myc, cyclin B1 and pro caspase expression levels were evaluated by western blot analysis. We found that expression of c-Myc was highly expressed in the SW13 cells, whereas the protein was undetectable in the H295R cells. Different doses of paclitaxel were required in the two ACC cell line to induce a block in the G2 phase, characterized by increased cyclin B1 levels and to induce apoptosis by pro-caspase-3 activation. Interestingly, the silencing of C-MYC mRNA prevented paclitaxel induced apoptosis in SW13 cells, whereas in the H295R cells the overexpression of C-MYC rendered the cells more prone to growth inhibition after paclitaxel exposure. The present study directly demonstrates that C-MYC plays a central role in controlling proliferation in ACC cells after paclitaxel treatment and that c-Myc could be considered as a marker for predicting response to chemotherapeutic agents in ACC cell lines.