Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage.

This unit describes immunocytochemical detection of histone H2AX phosphorylated on Ser-139 (γH2AX) to reveal DNA damage, particularly when the damage involves the presence of DNA double-strand breaks (DSBs). These breaks often result from DNA damage induced by ionizing radiation or by treatment with anticancer drugs such as DNA topoisomerase inhibitors. Furthermore, DSBs are generated in the course of DNA fragmentation during apoptosis.

Concurrent detection of lysosome and tissue transglutaminase activation in relation to cell cycle position during apoptosis induced by different anticancer drugs.

Described is the new cytometric approach do detect either stimulation or a collapse of lysosomal proton pump (lysosomes rupture) combined with activation of transglutaminase 2 (TG2) during induction of apoptosis. Apoptosis of human lymphoblastoid TK6 cells was induced by combination of 2-deoxyglucose with the isoquinoline alkaloid berberine, by DNA topoisomerase I inhibitor camptothecin, its analog topotecan, topoisomerase II inhibitors etoposide or mitoxantrone, as well as by the cytotoxic anticancer ribonuclease ranpirnase (onconase). Activity of the proton pump of lysosomes was assessed by measuring entrapment and accumulation of the basic fluorochrome acridine orange (AO) resulting in its metachromatic red luminescence (F ) within these organelles.

Upregulation of PD‑L1 expression by resveratrol and piceatannol in breast and colorectal cancer cells occurs via HDAC3/p300‑mediated NF‑κB signaling.

Programmed cell death ligand 1 (PD‑L1) expressed in cancer cells interacting with its receptor programmed cell death 1 (PD‑1) expressed in immune cells represents a regulatory axis linked to the suppression and evasion of host immune functions. The blockade of PD‑1/PD‑L1 interaction using monoclonal antibodies has emerged as an effective therapy for several solid tumors; however, durable response has been observed in a subset of patients with PD‑L1-positive tumors. Thus, the understanding of the mechanisms responsible for the expression of PD‑L1 in tumor cells may help to improve the response to PD‑L1 blockade therapies.

ATM Activation and H2AX Phosphorylation Induced by Genotoxic Agents Assessed by Flow- and Laser Scanning Cytometry.

Activation of Ataxia Telangiectasia Mediated protein kinase (ATM) by its phosphorylation on serine 1981 and phosphorylation of histone H2AX on serine 139 (γH2AX) are the key events reporting DNA damage, primarily formation of DNA double strand breaks. These events are detected immunocytochemically in individual cells using phospho-specific Abs. The protocols are presented that describe the methodology of immunofluorescent labeling of cells in conjunction with specific staining of cellular DNA.