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Flow cytometric studies on actin polymerization in PMN cells from chronic myeloid leukemia (CML) patients
N R Naik1, A N Bhisey, S H Advani
1Cancer Research Institute, Tata Memorial Centre, Parel, Bombay, India.
Abstract:
Studies in our laboratory have shown that polymorphonuclear leucocytes (PMNL) from chronic myeloid leukemia (CML) patients are defective in chemotaxis towards a synthetic peptide, n-formyl-methionyl-leucyl-phenylalanine (FMLP), during the active phases of the disease and in remission. Actin plays a major role in cellular movements and binding of chemo-attractant to cells induces polymerization of G-actin to F-actin. We have, therefore, compared polymerization of actin in FMLP stimulated PMNL from CML patients with those from normal subjects by fluorescence microscopy and flow cytometry, using F-actin specific probe, NBD-phallacidin. Our results show that binding of FMLP to normal PMNL induces rapid conversion of G-actin to F-actin followed by depolymerization to some extent. In CML PMNL, such a biphasic response is not seen. Conversion of G-actin to F-actin is slower and F-actin content is significantly lower than that in normal PMNL. Moreover, organization of F-actin is different in CML PMNL as compared to that in normal PMNL.
Insights
Polymorphonuclear leucocytes (PMNL) from chronic myeloid leukemia (CML) patients show defective actin polymerization in response to chemoattractants. This actin dysfunction impairs cell movement in CML patients.
Area of Science:
- Hematology
- Cell Biology
- Biochemistry
Background:
- Polymorphonuclear leucocytes (PMNL) are crucial for immune response.
- Defective PMNL chemotaxis is observed in chronic myeloid leukemia (CML).
- Actin polymerization is essential for cellular motility and response to chemoattractants like n-formyl-methionyl-leucyl-phenylalanine (FMLP).
Purpose of the Study:
- To investigate actin polymerization dynamics in FMLP-stimulated PMNL from CML patients compared to normal subjects.
- To understand the role of actin cytoskeleton alterations in CML-related PMNL dysfunction.
Main Methods:
- Utilized fluorescence microscopy and flow cytometry.
- Employed NBD-phallacidin, an F-actin specific probe.
- Compared actin polymerization in PMNL from CML patients and healthy controls upon FMLP stimulation.
Main Results:
- Normal PMNL exhibit a biphasic actin polymerization response (G-actin to F-actin conversion followed by partial depolymerization) upon FMLP stimulation.
- CML PMNL display a slower G-actin to F-actin conversion with significantly lower F-actin content.
- Altered F-actin organization was observed in CML PMNL compared to normal PMNL.
Conclusions:
- CML PMNL demonstrate impaired actin polymerization and altered F-actin organization in response to chemoattractants.
- These actin cytoskeleton abnormalities contribute to the defective chemotaxis observed in CML.
- Targeting actin dynamics may offer therapeutic potential for CML-related immune dysfunction.