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Cytosolic calcium pre-elevation amplifies agonist-induced calcium release in human leukaemic HL-60 cells.

ABSTRACT: Histamine, ATP, and two microsomal Ca(2+)-pump inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), were able to release intracellular Ca2+ in human leukaemic HL-60 cells. The relationships between the agonist-, TG- and CPA-sensitive Ca2+ pools were investigated with optimal concentrations of these agents in Ca(2+)-free medium. CPA failed to release Ca2+ after the Ca2+ stores of the cells had been discharged by TG, and vice versa, suggesting that the TG- and CPA-sensitive pools exactly overlap. Using this protocol, it was further demonstrated that (a) histamine and ATP utilized the same agonist-sensitive pool, and (b) the CPA- or TG-sensitive pool was much larger than, and encompassed, the agonist-sensitive pool. Although optimal (30 microM) CPA treatment for 5 min totally emptied the agonist-sensitive pool, a brief exposure (1.5 min) to a sub-optimal concentration (3 microM) of CPA, which only slightly raised cytosolic free Ca2+ concentration ([Ca2+]i), substantially enhanced subsequent agonist-induced Ca2+ release. Brief pretreatments with sub-optimal concentrations of TG or ionomycin, which caused moderate [Ca2+]i elevation, also caused such enhancement. However, sub-optimal CPA pretreatment had no prominent effect on Ca2+ release, which was InsP3-independent: it did not enhance TG-induced Ca2+ release, and only relatively weakly augmented ionomycin-induced Ca2+ release. Our results represent a novel observation showing that low concentrations of CPA, TG and ionomycin can potentiate subsequent agonist-induced Ca2+ release, and suggest that a 'priming' moderate [Ca2+]i elevation can amplify subsequent InsP3-dependent Ca2+ release in HL-60 cells.

SUBMITTER: Leung YM

PROVIDER: S-EPMC1137194 | biostudies-other | 1994 Aug

REPOSITORIES: biostudies-other

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Project description:Tumour microenvironments are hallmarked in many cancer types. In haematological malignancies, bone marrow (BM) mesenchymal stromal cells (MSC) protect malignant cells from drug-induced cytotoxicity. However, less is known about malignant impact on supportive stroma. Notably, it is unknown whether these interactions alter long-term genotoxic damage in either direction. The nucleoside analogue cytarabine (ara-C), common in haematological therapies, remains the most effective agent for acute myeloid leukaemia, yet one-third of patients develop resistance. This study aimed to evaluate the bidirectional effect of MSC and malignant cell co-culture on ara-C genotoxicity modulation. Primary MSC, isolated from patient BM aspirates for haematological investigations, and malignant haematopoietic cells (leukaemic HL-60) were co-cultured using trans-well inserts, prior to treatment with physiological dose ara-C. Co-culture genotoxic effects were assessed by micronucleus and alkaline comet assays. Patient BM cells from chemotherapy-treated patients had reduced ex vivo survival (P = 0.0049) and increased genotoxicity (P = 0.3172) than untreated patients. It was shown for the first time that HL-60 were protected by MSC from ara-C-induced genotoxicity, with reduced MN incidence in co-culture as compared to mono-culture (P = 0.0068). Comet tail intensity also significantly increased in ara-C-treated MSC with HL-60 influence (P = 0.0308). MSC sensitisation to ara-C genotoxicity was also demonstrated following co-culture with HL60 (P = 0.0116), which showed significantly greater sensitisation when MSC-HL-60 co-cultures were exposed to ara-C (P = 0.0409). This study shows for the first time that malignant HSC and MSC bidirectionally modulate genotoxicity, providing grounding for future research identifying mechanisms of altered genotoxicity in leukaemic microenvironments. MSC retain long-term genotoxic and functional damage following chemotherapy exposure. Understanding the interactions perpetuating such damage may inform modifications to reduce therapy-related complications, such as secondary malignancies and BM failure.

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Cytosolic calcium pre-elevation amplifies agonist-induced calcium release in human leukaemic HL-60 cells.

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