Low and high molecular weight poly(L-lysine)s/poly(L-lysine)-DNA complexes initiate mitochondrial-mediated apoptosis differently.
Poly(L-lysine)s, PLLs, are commonly used for DNA compaction and cell transfection. We report that, although PLLs of low (2.9 kDa), L-PLL, and high (27.4 kDa), H-PLL, Mw in free form and DNA-complexed cannot only cause rapid plasma membrane damage in human cell lines, phosphatidylserine "scrambling" and loss of membrane integrity, but later (24 h) initiate stress-induced cell death via mitochondrial permeabilization without the involvement of processed caspase-2. Mitochondrially mediated apoptosis was confirmed by detection of cytochrome c (Cyt c) release, activation of caspases-9 and -3, and subsequent changes in mitochondrial membrane potential. Plasma membrane damage and apoptosis were most prominent with H-PLL. Cytoplasmic level of Cyt c was more elevated following H-PLL treatment, but unlike L-PLL case, inhibition of Bax channel-forming activity reduced the extent of Cyt c release from mitochondria by half. Inhibition of Bax channel-forming activity had no modulatory effect on L-PLL-mediated Cyt c release. Further, functional studies of isolated mitochondria indicate that H-PLL, but not L-PLL, can directly induce Cyt c release, membrane depolarization, and a progressive decline in the rate of uncoupled respiration. Combined, our data suggest that H-PLL and L-PLL are capable of initiating mitochondrially mediated apoptosis differently. The observed PLL-mediated late-phase apoptosis may provide an explanation for previously reported transient gene expression associated with PLL-based transfection vectors. The importance of our data in relation to design of novel and safer cationic non-viral vectors for human gene therapy is discussed.
Symonds
Peter
P
Cancer Research-UK, Tumour Cytokine Biology Group, University Hospital, Nottingham NG7 2UH, UK.
Murray
J Clifford
JC
Hunter
A Christy
AC
Debska
Grazyna
G
Szewczyk
Adam
A
Moghimi
S Moein
SM
D017209:Apoptosis; D001692:Biological Transport; D020169:Caspases / Q000032:analysis; D045304:Cytochromes c / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D010539:Permeability; D010718:Phosphatidylserines / Q000378:metabolism; D011107:Polylysine / Q000737:chemistry / Q000494:pharmacology*
D010718:Phosphatidylserines; D011107:Polylysine; D045304:Cytochromes c; D004247:DNA; D020169:Caspases