We studied the effectiveness of trilysine (Lys3), tetralysine (Lys4), pentalysine (Lys5), and poly-l-lysine (PLL) (MW 50000) on lambda-DNA nanoparticle formation and characterized the size, shape, and stability of nanoparticles. Light scattering experiments showed EC50 (lysine concentration at 50% DNA compaction) values of approximately 0.0036, 2, and 20 micromol/L, respectively, for PLL, Lys5, and Lys4 at 10 mM [Na+]. Plots of log EC50 versus log [Na+] showed positive slopes of 1.09 and 1.7, respectively, for Lys4 and Lys5 and a negative slope of -0.1 for PLL. Hydrodynamic radii of oligolysine condensed particles increased (48-173 nm) with increasing [Na+], whereas no significant change occurred to nanoparticles formed with PLL. There was an increase in the size of nanoparticles formed with Lys5 at >40 degrees C, whereas no such change occurred with PLL. The DNA melting temperature increased with oligolysine concentration. These results indicate distinct differences in the mechanism(s) by which oligolysines and PLL provoke DNA condensation to nanoparticles.
Beclin 1 is an essential mediator of autophagy and a regulator of cell growth and cell death. We examined the effect of Beclin 1 overexpression on the action of estradiol (E 2 ) and two antiestrogens, raloxifene and 4-hydroxytamoxifen, in estrogen receptor A (ERA)-positive MCF-7 breast cancer cells. [3 H]-thymidine incorporation studies showed that Beclin 1-overexpressing cells (MCF-7.beclin) had a lower proliferative response to E 2 compared with cells transfected with vector control (MCF-7.control). There was only a 35% increase in [ 3 H]-thymidine incorporation, after 24 hours of E 2 treatment of MCF-7.beclin cells compared with untreated cells, whereas this increase was 2-fold for MCF-7.control cells. E 2 -induced changes in the expression of early-response genes were examined by real-time quantitiative PCR. There were significant differences in the pattern of expression of E 2 -induced genes c-myc, c-fos, Erg-1, and Nur77 between MCF-7.beclin and MCF-7.control cells two hours after treatment. Although E 2 -induced growth of MCF-7.control cells was completely inhibited by 500 nmol/L raloxifene or 500 nmol/L 4-hydroxytamoxifen, these concentrations of antiestrogens had no significant effect on the growth of MCF-7.beclin cells. Confocal microscopic and coimmunoprecipitation studies showed evidence for colocalization and association of Beclin 1 and ERA. In addition, E 2 caused a decrease in Akt phosphorylation in MCF-7.beclin cells, compared with a 3-fold increase in MCF-7 cells, five minutes after treatment. These results indicate that Beclin 1 can down-regulate estrogenic signaling and growth response, and contribute to the development of antiestrogen resistance. This observation might be useful to define and overcome antiestrogen resistance of breast cancer.
Polyamines are essential molecules supporting the structure, conformation, and function of nucleic acids and proteins. We studied stereoisomers of alpha,alpha'-dimethylated spermine [(R,R)-Me(2)Spm, (S,S)-Me(2)Spm, (R,S)-Me(2)Spm] for their ability to provoke DNA condensation and protect DNA from damage. (R,R)- and (R,S)-Me(2)Spm displayed more efficient condensing ability than spermine, with significantly lower EC(50) (concentration for 50% compaction) values (p < or = 0.01). However, spermine exerted slightly more duplex stabilization than Me(2)Spm. Condensation resulted in nanoparticles with hydrodynamic radii between 39.6 and 48.4 nm, and electron microscopy showed the presence of toroids and spheroids. Natural polyamines and stereoisomers of Me(2)Spm protected DNA against DNase digestion and oxidative stress in vitro and against etoposide and oxidative stress in DU145 cells but afforded little protection against UV-C irradiation. Our findings indicate that Me(2)Spm stereoisomers are efficient DNA packaging agents with potential applications in gene delivery. Our study also reveals stereospecificity in DNA interaction and protection against cellular stress.
BE-3-3-3-3 (1,15-(ethylamino)4,8,12-triazapentadecane) is a bis(ethyl)polyamine analogue under investigation as a therapeutic agent for breast cancer. Since estradiol (E(2)) is a critical regulatory molecule in the growth of breast cancer, we examined the effect of BE-3-3-3-3 on estrogen receptor α (ERα) positive MCF-7 cells in the presence and absence of E(2). In the presence of E(2), a concentration-dependent decrease in DNA synthesis was observed using [(3)H]-thymidine incorporation assay. In the absence of E(2), low concentrations (2.5-10 μM) of BE-3-3-3-3 increased [(3)H]-thymidine incorporation at 24 and 48 h. BE-3-3-3-3 induced the expression of early response genes, c-myc and c-fos, in the absence of E(2), but not in its presence, as determined by real-time quantitative polymerase chain reaction (qPCR). BE-3-3-3-3 had no significant effect on these genes in an ERα-negative cell line, MDA-MB-231. Chromatin immunoprecipitation assay demonstrated enhanced promoter occupation by either E(2) or BE-3-3-3-3 of an estrogen-responsive gene pS2/Tff1 by ERα and its co-activator, steroid receptor co-activator 3 (SRC-3). Confocal microscopy of BE-3-3-3-3-treated cells revealed membrane localization of ERα, similar to that induced by E(2). The failure of BE-3-3-3-3 to inhibit cell proliferation was associated with autophagic vacuole formation, and the induction of Beclin 1 and MAP LC3 II. These results indicate a differential effect of BE-3-3-3-3 on MCF-7 cells in the absence and presence of E(2), and suggest that pre-clinical and clinical development of polyamine analogues might require special precautions and selection of sensitive subpopulation of patients.
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