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Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

He Q, Sun X, Chu C, Jiang Q, Zhu H, He Y, Yue T, Wang R, Lei P, Shen G - PLoS ONE (2015)

Bottom Line: Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis.The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles.This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.

ABSTRACT
The endocytosis of transferrin receptor (TfR) has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP)-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

No MeSH data available.


Related in: MedlinePlus

Validation of the hTfR-EGFP specificity.(A) Confocal imaging (4°C) and (B) flow cytometry studies on CHO and CHOvec(hTfR-) cells, CHO-hTfR (hTfR+) cells, HepG2 (hTfR+-wt) cells demonstrated the hTfR targeting of mAb and Tf. (B) Upper: representative FCM pictures were shown. Lower: The bar graph represented FCM analysis of the percentage of PE positive cells. Mean values ± standard deviation (SD), n = 3; P values were calculated on the basis of SPSS 17.0 statistical software (NS, not significant).
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pone.0122452.g004: Validation of the hTfR-EGFP specificity.(A) Confocal imaging (4°C) and (B) flow cytometry studies on CHO and CHOvec(hTfR-) cells, CHO-hTfR (hTfR+) cells, HepG2 (hTfR+-wt) cells demonstrated the hTfR targeting of mAb and Tf. (B) Upper: representative FCM pictures were shown. Lower: The bar graph represented FCM analysis of the percentage of PE positive cells. Mean values ± standard deviation (SD), n = 3; P values were calculated on the basis of SPSS 17.0 statistical software (NS, not significant).

Mentions: Next, we examined whether this binding was indeed hTfR-specific. For this purpose, vector control CHOvec cell line was set as a negative control (hTfR-) and HepG2 cells as positive control (hTfR+) for comparison with results obtained with the CHO-hTfR cells. Confocal imaging studies showed binding of mAb with CHO-hTfR(hTfR+) cells and HepG2 cells but not with CHOvec(hTfR-) cells (Fig 4A). This result also supported the western blot analysis (Fig 2B) suggesting that no detectable amount of free hTfR was present in hTfR-EGFP expressing cells. HepG2 cells showed a similar distribution of hTfR immunoreactivity, whereas no green fluorescence was detected (Fig 4A), indicating the specificity of the detection of hTfR-EGFP fluorescence in our system.


Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

He Q, Sun X, Chu C, Jiang Q, Zhu H, He Y, Yue T, Wang R, Lei P, Shen G - PLoS ONE (2015)

Validation of the hTfR-EGFP specificity.(A) Confocal imaging (4°C) and (B) flow cytometry studies on CHO and CHOvec(hTfR-) cells, CHO-hTfR (hTfR+) cells, HepG2 (hTfR+-wt) cells demonstrated the hTfR targeting of mAb and Tf. (B) Upper: representative FCM pictures were shown. Lower: The bar graph represented FCM analysis of the percentage of PE positive cells. Mean values ± standard deviation (SD), n = 3; P values were calculated on the basis of SPSS 17.0 statistical software (NS, not significant).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372551&req=5

pone.0122452.g004: Validation of the hTfR-EGFP specificity.(A) Confocal imaging (4°C) and (B) flow cytometry studies on CHO and CHOvec(hTfR-) cells, CHO-hTfR (hTfR+) cells, HepG2 (hTfR+-wt) cells demonstrated the hTfR targeting of mAb and Tf. (B) Upper: representative FCM pictures were shown. Lower: The bar graph represented FCM analysis of the percentage of PE positive cells. Mean values ± standard deviation (SD), n = 3; P values were calculated on the basis of SPSS 17.0 statistical software (NS, not significant).
Mentions: Next, we examined whether this binding was indeed hTfR-specific. For this purpose, vector control CHOvec cell line was set as a negative control (hTfR-) and HepG2 cells as positive control (hTfR+) for comparison with results obtained with the CHO-hTfR cells. Confocal imaging studies showed binding of mAb with CHO-hTfR(hTfR+) cells and HepG2 cells but not with CHOvec(hTfR-) cells (Fig 4A). This result also supported the western blot analysis (Fig 2B) suggesting that no detectable amount of free hTfR was present in hTfR-EGFP expressing cells. HepG2 cells showed a similar distribution of hTfR immunoreactivity, whereas no green fluorescence was detected (Fig 4A), indicating the specificity of the detection of hTfR-EGFP fluorescence in our system.

Bottom Line: Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis.The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles.This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.

ABSTRACT
The endocytosis of transferrin receptor (TfR) has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP)-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

No MeSH data available.


Related in: MedlinePlus