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Published on: March 25, 2016
Flow cytometric evaluation of fas expression in relation to response and resistance to anthracyclines in leukemic
G Labroille1, P Dumain, F Lacombe
1Laboratoire d'hématologie, Hôpital du Haut-Lévêque, Pessac, France.
Background:
Cell chemosensitivity to cytotoxic drugs has been attributed to their ability to trigger apoptosis. The emergence of resistance in drug-exposed cells is often characterized by the appearance of drug efflux mechanisms including P-gp transport. Nevertheless, mdr1 expression may coexist with other resistance features, in particular those interfering with apoptotic signaling pathways.
Methods:
Leukemic cell lines cultured in a progressively toxic environment were analyzed for Fas and P-gp expression by immunostaining and flow cytometry. Their mdr1 mRNA expression level was determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and their apoptotic response was microscopically evaluated. Activation of the Fas pathway was obtained by cross-linking the Fas receptor with the 7C11 anti-Fas agonist.
Results:
We demonstrate a dose-dependent Fas overexpression after short-term (18 h) incubation with daunorubicin. The subsequent sensitization to Fas activators led to a significant increase in the apoptotic response induced by 7C11. After long-term exposure to daunorubicin and acquisition of drug resistance, expression of P-gp was accompanied by a decrease in the number of Fas sites at the cell surface with a correlated desensitization to Fas-induced apoptosis. Additional alterations in the Fas signaling pathway can also be hypothesized in the most resistant Jurkat cell line.
Conclusions:
The induction of Fas expression could be one of the mechanisms of action of chemotoxic drugs and thus might enhance the cell susceptibility to Fas-mediated apoptosis. On the contrary, the emergence of the multidrug resistance phenotype is associated with a down-regulation of Fas expression and possible defects in the Fas signaling pathway.
Insights
Chemotherapy can increase cancer cell sensitivity to apoptosis by upregulating Fas expression. However, multidrug resistance development leads to decreased Fas expression, impairing apoptosis.
Area of Science:
- Cell biology
- Cancer research
- Molecular oncology
Background:
- Cell chemosensitivity to cytotoxic drugs is linked to apoptosis induction.
- Drug resistance often involves P-glycoprotein (P-gp) efflux pumps.
- Resistance mechanisms can also involve disruptions in apoptotic signaling pathways.
Purpose of the Study:
- To investigate the relationship between daunorubicin exposure, Fas expression, and apoptosis in leukemic cell lines.
- To explore how multidrug resistance affects Fas-mediated apoptosis signaling.
Main Methods:
- Leukemic cell lines were cultured in a toxic environment with increasing daunorubicin concentrations.
- Fas and P-gp expression were analyzed using immunostaining and flow cytometry.
- mRNA levels (mdr1) and apoptotic responses were assessed via RT-PCR and microscopy.
- Fas pathway activation was induced using an anti-Fas agonist (7C11).
Main Results:
- Short-term daunorubicin exposure caused dose-dependent Fas overexpression and enhanced apoptosis upon Fas activation.
- Long-term exposure led to drug resistance, P-gp expression, and decreased Fas sites, resulting in Fas-induced apoptosis desensitization.
- Resistant Jurkat cells showed further alterations in the Fas signaling pathway.
Conclusions:
- Fas expression induction may be a mechanism for chemotoxic drugs to enhance apoptosis susceptibility.
- Multidrug resistance development is associated with reduced Fas expression and potential defects in Fas signaling.
- Understanding these pathways is crucial for overcoming chemoresistance.
