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

3E10 scFv localizes to tumor cell nuclei in vivo.Immunodeficient mice bearing subcutaneous U87 human glioma xenografts were treated by intraperitoneal injection of control buffer or 3E10 scFv. Mice were sacrificed 4 or 24 hours after treatment, and tumors and select normal tissues were immunostained for the presence of 3E10 scFv. Brown nuclear stain indicates presence of 3E10 scFv, while blue nuclear stain is negative. (A) Four hours after treatment 3E10 scFv was detected in the nuclei of the U87 tumor cells but was not detected in tissues of major organs including heart, kidney, skeletal muscle, and liver. These results are consistent with preferential uptake of 3E10 scFv into tumors. (B) Twenty-four hours after treatment 3E10 scFv was still detectable in the tumors, demonstrating the stability of the uptake into tumor nuclei.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: 3E10 scFv localizes to tumor cell nuclei in vivo.Immunodeficient mice bearing subcutaneous U87 human glioma xenografts were treated by intraperitoneal injection of control buffer or 3E10 scFv. Mice were sacrificed 4 or 24 hours after treatment, and tumors and select normal tissues were immunostained for the presence of 3E10 scFv. Brown nuclear stain indicates presence of 3E10 scFv, while blue nuclear stain is negative. (A) Four hours after treatment 3E10 scFv was detected in the nuclei of the U87 tumor cells but was not detected in tissues of major organs including heart, kidney, skeletal muscle, and liver. These results are consistent with preferential uptake of 3E10 scFv into tumors. (B) Twenty-four hours after treatment 3E10 scFv was still detectable in the tumors, demonstrating the stability of the uptake into tumor nuclei.

Mentions: As described above, 3E10 scFv has potential applications in cancer therapy both as a delivery vehicle and as an inhibitor of DNA repair. After confirming that 3E10 scFv penetrates cell nuclei most efficiently in the presence of extracellular DNA, we hypothesized that the fragment would preferentially localize into the nuclei of tumor cells in vivo due to an expected higher concentration of extracellular DNA in the tumor vicinity released from dead cells in regions of tumor ischemia and necrosis. To test this, subcutaneous U87 human glioma xenografts were generated in immunodeficient mice, and once tumors grew to size of ~100 mm3 mice were treated with intraperitoneal injection of control buffer or 3E10 scFv. Mice were then sacrificed 4 or 24 hours after treatment, and tumors and select normal tissues were immunostained for the presence of 3E10 scFv. Four hours after treatment 3E10 scFv was not detected in the cell nuclei of normal tissues including heart, kidney, skeletal muscle, and liver. By contrast, cell nuclei in the tumor xenografts stained positive for presence of 3E10 scFv (Fig. 3A). 3E10 scFv was also detected in the tumors 24 hours after treatment, demonstrating the stability of the uptake into tumor nuclei (Fig. 3B). These results are consistent with preferential uptake of 3E10 scFv into tumors.


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 localizes to tumor cell nuclei in vivo.Immunodeficient mice bearing subcutaneous U87 human glioma xenografts were treated by intraperitoneal injection of control buffer or 3E10 scFv. Mice were sacrificed 4 or 24 hours after treatment, and tumors and select normal tissues were immunostained for the presence of 3E10 scFv. Brown nuclear stain indicates presence of 3E10 scFv, while blue nuclear stain is negative. (A) Four hours after treatment 3E10 scFv was detected in the nuclei of the U87 tumor cells but was not detected in tissues of major organs including heart, kidney, skeletal muscle, and liver. These results are consistent with preferential uptake of 3E10 scFv into tumors. (B) Twenty-four hours after treatment 3E10 scFv was still detectable in the tumors, demonstrating the stability of the uptake into tumor nuclei.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: 3E10 scFv localizes to tumor cell nuclei in vivo.Immunodeficient mice bearing subcutaneous U87 human glioma xenografts were treated by intraperitoneal injection of control buffer or 3E10 scFv. Mice were sacrificed 4 or 24 hours after treatment, and tumors and select normal tissues were immunostained for the presence of 3E10 scFv. Brown nuclear stain indicates presence of 3E10 scFv, while blue nuclear stain is negative. (A) Four hours after treatment 3E10 scFv was detected in the nuclei of the U87 tumor cells but was not detected in tissues of major organs including heart, kidney, skeletal muscle, and liver. These results are consistent with preferential uptake of 3E10 scFv into tumors. (B) Twenty-four hours after treatment 3E10 scFv was still detectable in the tumors, demonstrating the stability of the uptake into tumor nuclei.
Mentions: As described above, 3E10 scFv has potential applications in cancer therapy both as a delivery vehicle and as an inhibitor of DNA repair. After confirming that 3E10 scFv penetrates cell nuclei most efficiently in the presence of extracellular DNA, we hypothesized that the fragment would preferentially localize into the nuclei of tumor cells in vivo due to an expected higher concentration of extracellular DNA in the tumor vicinity released from dead cells in regions of tumor ischemia and necrosis. To test this, subcutaneous U87 human glioma xenografts were generated in immunodeficient mice, and once tumors grew to size of ~100 mm3 mice were treated with intraperitoneal injection of control buffer or 3E10 scFv. Mice were then sacrificed 4 or 24 hours after treatment, and tumors and select normal tissues were immunostained for the presence of 3E10 scFv. Four hours after treatment 3E10 scFv was not detected in the cell nuclei of normal tissues including heart, kidney, skeletal muscle, and liver. By contrast, cell nuclei in the tumor xenografts stained positive for presence of 3E10 scFv (Fig. 3A). 3E10 scFv was also detected in the tumors 24 hours after treatment, demonstrating the stability of the uptake into tumor nuclei (Fig. 3B). These results are consistent with preferential uptake of 3E10 scFv into tumors.

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