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DNA-dependent targeting of cell nuclei by a lupus autoantibody.

Weisbart RH, Chan G, Jordaan G, Noble PW, Liu Y, Glazer PM, Nishimura RN, Hansen JE - Sci Rep (2015)

Bottom Line: A greater understanding of the mechanism by which 3E10 penetrates cell nuclei is needed to help determine the scope of its potential therapeutic applications.Here we show that the presence of extracellular DNA significantly enhances the nuclear uptake of 3E10 scFv.In addition, we find that 3E10 scFv preferentially localizes into tumor cell nuclei in vivo, likely due to increased DNA in the local environment released from ischemic and necrotic regions of tumor.

View Article: PubMed Central - PubMed

Affiliation: Department of Research, Veterans Affairs Greater Los Angeles Healthcare System, Sepulveda, CA 91343.

ABSTRACT
A nuclear-penetrating lupus anti-DNA autoantibody, 3E10, has been found to inhibit DNA repair and selectively kill certain cancer cells that are highly vulnerable to DNA damage. In addition, a 3E10 single chain variable fragment (scFv) has been developed for use as a delivery vehicle to carry therapeutic cargo proteins into cell nuclei. A greater understanding of the mechanism by which 3E10 penetrates cell nuclei is needed to help determine the scope of its potential therapeutic applications. Here we show that the presence of extracellular DNA significantly enhances the nuclear uptake of 3E10 scFv. In addition, we find that 3E10 scFv preferentially localizes into tumor cell nuclei in vivo, likely due to increased DNA in the local environment released from ischemic and necrotic regions of tumor. These data provide insight into the mechanism of nuclear penetration by 3E10 and demonstrate the potential for use of 3E10 in therapeutic approaches to diseases ranging from malignancy to ischemic conditions such as stroke.

No MeSH data available.


Related in: MedlinePlus

3E10 scFv penetrates most efficiently into living cells surrounding a dead cell.GM02605 fibroblasts were washed with serum free media and then treated with 10 μM 3E10 scFv for one hour, followed by anti-Myc immunostaining to detect nuclear penetration by 3E10 scFv. Nuclear penetration by 3E10 scFv was restricted to cells in close proximity to what morphologically appears to be a dead cell, suggesting that a factor released by dead cells promotes nuclear uptake of the fragment.
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f1: 3E10 scFv penetrates most efficiently into living cells surrounding a dead cell.GM02605 fibroblasts were washed with serum free media and then treated with 10 μM 3E10 scFv for one hour, followed by anti-Myc immunostaining to detect nuclear penetration by 3E10 scFv. Nuclear penetration by 3E10 scFv was restricted to cells in close proximity to what morphologically appears to be a dead cell, suggesting that a factor released by dead cells promotes nuclear uptake of the fragment.

Mentions: We sought to test the efficiency of nuclear penetration by 3E10 scFv into cells in the absence of extracellular DNA, and for these studies selected the GM02605 human fibroblast cell line because it maintains a high degree of viability (>99%) with minimal cell death even while maintained in culture for several days. With minimal cell death in culture the confounding effects of DNA released by dead cells are minimized. The GM02605 cells were washed with serum free media and then treated with 10 μM 3E10 scFv for one hour, after which cells were fixed and immunostained for presence of the fragment. Remarkably, 3E10 scFv did not penetrate into most cells. Instead, 3E10 scFv was found only in the nuclei of cells centered around what morphologically appeared to be rare dead cells, with a gradient effect observed with decreased amounts of intranuclear fragment detected with increasing distance from the central dead cell. A representative image demonstrating this effect is shown in Fig. 1. This observation was consistent with a factor released locally by dead cells enhancing nuclear penetration by 3E10 scFv into surrounding cells.


DNA-dependent targeting of cell nuclei by a lupus autoantibody.

Weisbart RH, Chan G, Jordaan G, Noble PW, Liu Y, Glazer PM, Nishimura RN, Hansen JE - Sci Rep (2015)

3E10 scFv penetrates most efficiently into living cells surrounding a dead cell.GM02605 fibroblasts were washed with serum free media and then treated with 10 μM 3E10 scFv for one hour, followed by anti-Myc immunostaining to detect nuclear penetration by 3E10 scFv. Nuclear penetration by 3E10 scFv was restricted to cells in close proximity to what morphologically appears to be a dead cell, suggesting that a factor released by dead cells promotes nuclear uptake of the fragment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496662&req=5

f1: 3E10 scFv penetrates most efficiently into living cells surrounding a dead cell.GM02605 fibroblasts were washed with serum free media and then treated with 10 μM 3E10 scFv for one hour, followed by anti-Myc immunostaining to detect nuclear penetration by 3E10 scFv. Nuclear penetration by 3E10 scFv was restricted to cells in close proximity to what morphologically appears to be a dead cell, suggesting that a factor released by dead cells promotes nuclear uptake of the fragment.
Mentions: We sought to test the efficiency of nuclear penetration by 3E10 scFv into cells in the absence of extracellular DNA, and for these studies selected the GM02605 human fibroblast cell line because it maintains a high degree of viability (>99%) with minimal cell death even while maintained in culture for several days. With minimal cell death in culture the confounding effects of DNA released by dead cells are minimized. The GM02605 cells were washed with serum free media and then treated with 10 μM 3E10 scFv for one hour, after which cells were fixed and immunostained for presence of the fragment. Remarkably, 3E10 scFv did not penetrate into most cells. Instead, 3E10 scFv was found only in the nuclei of cells centered around what morphologically appeared to be rare dead cells, with a gradient effect observed with decreased amounts of intranuclear fragment detected with increasing distance from the central dead cell. A representative image demonstrating this effect is shown in Fig. 1. This observation was consistent with a factor released locally by dead cells enhancing nuclear penetration by 3E10 scFv into surrounding cells.

Bottom Line: A greater understanding of the mechanism by which 3E10 penetrates cell nuclei is needed to help determine the scope of its potential therapeutic applications.Here we show that the presence of extracellular DNA significantly enhances the nuclear uptake of 3E10 scFv.In addition, we find that 3E10 scFv preferentially localizes into tumor cell nuclei in vivo, likely due to increased DNA in the local environment released from ischemic and necrotic regions of tumor.

View Article: PubMed Central - PubMed

Affiliation: Department of Research, Veterans Affairs Greater Los Angeles Healthcare System, Sepulveda, CA 91343.

ABSTRACT
A nuclear-penetrating lupus anti-DNA autoantibody, 3E10, has been found to inhibit DNA repair and selectively kill certain cancer cells that are highly vulnerable to DNA damage. In addition, a 3E10 single chain variable fragment (scFv) has been developed for use as a delivery vehicle to carry therapeutic cargo proteins into cell nuclei. A greater understanding of the mechanism by which 3E10 penetrates cell nuclei is needed to help determine the scope of its potential therapeutic applications. Here we show that the presence of extracellular DNA significantly enhances the nuclear uptake of 3E10 scFv. In addition, we find that 3E10 scFv preferentially localizes into tumor cell nuclei in vivo, likely due to increased DNA in the local environment released from ischemic and necrotic regions of tumor. These data provide insight into the mechanism of nuclear penetration by 3E10 and demonstrate the potential for use of 3E10 in therapeutic approaches to diseases ranging from malignancy to ischemic conditions such as stroke.

No MeSH data available.


Related in: MedlinePlus