Limits...
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

Extracellular DNA facilitates penetration of 3E10 scFv into cell nuclei.GM02605 fibroblasts were washed with serum free media and then treated with control buffer alone or 10 μM 3E10 scFv in the presence of control buffer, cell lysate, DNA-depleted cell lysate, or purified DNA for one hour, followed by anti-Myc immunostaining to detect nuclear penetration by 3E10 scFv. Nuclear penetration into ~100% of the cells was only observed in the presence of cell lysate or purified DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496662&req=5

f2: Extracellular DNA facilitates penetration of 3E10 scFv into cell nuclei.GM02605 fibroblasts were washed with serum free media and then treated with control buffer alone or 10 μM 3E10 scFv in the presence of control buffer, cell lysate, DNA-depleted cell lysate, or purified DNA for one hour, followed by anti-Myc immunostaining to detect nuclear penetration by 3E10 scFv. Nuclear penetration into ~100% of the cells was only observed in the presence of cell lysate or purified DNA.

Mentions: To test the hypothesis that a factor released by dead cells enhances nuclear uptake of 3E10 scFv we compared the efficiency of nuclear penetration of the fragment into the GM02605 fibroblasts in the presence or absence of a cell lysate. As shown in Fig. 2, in the absence of the cell lysate minimal nuclear uptake of 3E10 scFv was observed. However, the addition of cell lysate facilitated nuclear penetration by 3E10 scFv into ~100% of the cells. These results further support the hypothesis that a factor released by dead cells and contained in cell lysate promotes nuclear uptake of 3E10 scFv.


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)

Extracellular DNA facilitates penetration of 3E10 scFv into cell nuclei.GM02605 fibroblasts were washed with serum free media and then treated with control buffer alone or 10 μM 3E10 scFv in the presence of control buffer, cell lysate, DNA-depleted cell lysate, or purified DNA for one hour, followed by anti-Myc immunostaining to detect nuclear penetration by 3E10 scFv. Nuclear penetration into ~100% of the cells was only observed in the presence of cell lysate or purified DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Extracellular DNA facilitates penetration of 3E10 scFv into cell nuclei.GM02605 fibroblasts were washed with serum free media and then treated with control buffer alone or 10 μM 3E10 scFv in the presence of control buffer, cell lysate, DNA-depleted cell lysate, or purified DNA for one hour, followed by anti-Myc immunostaining to detect nuclear penetration by 3E10 scFv. Nuclear penetration into ~100% of the cells was only observed in the presence of cell lysate or purified DNA.
Mentions: To test the hypothesis that a factor released by dead cells enhances nuclear uptake of 3E10 scFv we compared the efficiency of nuclear penetration of the fragment into the GM02605 fibroblasts in the presence or absence of a cell lysate. As shown in Fig. 2, in the absence of the cell lysate minimal nuclear uptake of 3E10 scFv was observed. However, the addition of cell lysate facilitated nuclear penetration by 3E10 scFv into ~100% of the cells. These results further support the hypothesis that a factor released by dead cells and contained in cell lysate promotes nuclear uptake of 3E10 scFv.

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