Bcl-2 overexpression protects photooxidative stress-induced apoptosis of photoreceptor cells via NF-κB preservation

Matthew J. Crawford, Raghu Krishnamoorthy, Victoria L. Rudick, Robert J. Collier, Michael Kapin, Bharat B. Aggarwal, Muayyad R. Al-Ubaidi, Neeraj Agarwal

Research output: Contribution to journalArticleResearchpeer-review

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Abstract

We recently showed that photooxidative stress on cultured photoreceptor cells results in down-modulation of NF-κB activity which then leads to apoptosis of cultured 661W photoreceptor cells. In an effort to further delineate the mechanism of photoreceptor cell death, we sought to determine the effects of Bcl-2 overexpression on cell survivability. Wild-type 661W cells were transfected with the plasmid construct pSFFV-neo-Bcl-2 and several clones were isolated. All clones demonstrated increased Bcl-2 mRNA and protein levels, with the B4 clone exhibiting the greatest enhancement. On exposure to visible light the B4 cells were protected from undergoing apoptosis when compared with the mock transfected cells, as ascertained by TUNEL apoptosis assay and formazan based estimation of cell viability. The Bcl-2 overexpressing cells also maintained a higher Bcl-2/Bax ratio, suggesting that this ratio is important in protection from photooxidative stress. Electrophoretic mobility shift assays for NF-κB demonstrated higher activity in both nuclear and cytosolic fractions of the B4 photoreceptors compared with the 661W wild-type cells at all light exposure time points. Furthermore, the findings of the gel shift assays were further supported by immunocytochemistry for NF-κB which revealed that protein levels of the RelA subunit of NF-κB were protected in the nucleus as well as in the cytoplasm of Bcl-2 overexpressing B4 cells exposed to light compared to the 661W cells. These results suggest that Bcl-2 overexpression protects NF-κB protein levels and activity in the nucleus, indicating that preservation of NF-κB binding activity in the nucleus may be essential for photoreceptor cells to survive photooxidative damage induced apoptosis. copy& 2001 Academic Press.

Original languageEnglish
Pages (from-to)1304-1312
Number of pages9
JournalBiochemical and Biophysical Research Communications
Volume281
Issue number5
DOIs
StatePublished - 1 Jan 2001

Fingerprint

Photoreceptor Cells
Apoptosis
Assays
Formazans
Electrophoretic mobility
Clone Cells
Cell death
Light
Proteins
Plasmids
Gels
Cells
Modulation
Messenger RNA
In Situ Nick-End Labeling
Electrophoretic Mobility Shift Assay
Cultured Cells
Cell Survival
Cytoplasm
Cell Death

Keywords

  • Bcl-2
  • Cell survival
  • Gene transfection
  • NF-κB
  • Photoreceptor cells
  • Retinal dystrophies

Cite this

Crawford, Matthew J. ; Krishnamoorthy, Raghu ; Rudick, Victoria L. ; Collier, Robert J. ; Kapin, Michael ; Aggarwal, Bharat B. ; Al-Ubaidi, Muayyad R. ; Agarwal, Neeraj. / Bcl-2 overexpression protects photooxidative stress-induced apoptosis of photoreceptor cells via NF-κB preservation. In: Biochemical and Biophysical Research Communications. 2001 ; Vol. 281, No. 5. pp. 1304-1312.
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abstract = "We recently showed that photooxidative stress on cultured photoreceptor cells results in down-modulation of NF-κB activity which then leads to apoptosis of cultured 661W photoreceptor cells. In an effort to further delineate the mechanism of photoreceptor cell death, we sought to determine the effects of Bcl-2 overexpression on cell survivability. Wild-type 661W cells were transfected with the plasmid construct pSFFV-neo-Bcl-2 and several clones were isolated. All clones demonstrated increased Bcl-2 mRNA and protein levels, with the B4 clone exhibiting the greatest enhancement. On exposure to visible light the B4 cells were protected from undergoing apoptosis when compared with the mock transfected cells, as ascertained by TUNEL apoptosis assay and formazan based estimation of cell viability. The Bcl-2 overexpressing cells also maintained a higher Bcl-2/Bax ratio, suggesting that this ratio is important in protection from photooxidative stress. Electrophoretic mobility shift assays for NF-κB demonstrated higher activity in both nuclear and cytosolic fractions of the B4 photoreceptors compared with the 661W wild-type cells at all light exposure time points. Furthermore, the findings of the gel shift assays were further supported by immunocytochemistry for NF-κB which revealed that protein levels of the RelA subunit of NF-κB were protected in the nucleus as well as in the cytoplasm of Bcl-2 overexpressing B4 cells exposed to light compared to the 661W cells. These results suggest that Bcl-2 overexpression protects NF-κB protein levels and activity in the nucleus, indicating that preservation of NF-κB binding activity in the nucleus may be essential for photoreceptor cells to survive photooxidative damage induced apoptosis. copy& 2001 Academic Press.",
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Bcl-2 overexpression protects photooxidative stress-induced apoptosis of photoreceptor cells via NF-κB preservation. / Crawford, Matthew J.; Krishnamoorthy, Raghu; Rudick, Victoria L.; Collier, Robert J.; Kapin, Michael; Aggarwal, Bharat B.; Al-Ubaidi, Muayyad R.; Agarwal, Neeraj.

In: Biochemical and Biophysical Research Communications, Vol. 281, No. 5, 01.01.2001, p. 1304-1312.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Bcl-2 overexpression protects photooxidative stress-induced apoptosis of photoreceptor cells via NF-κB preservation

AU - Crawford, Matthew J.

AU - Krishnamoorthy, Raghu

AU - Rudick, Victoria L.

AU - Collier, Robert J.

AU - Kapin, Michael

AU - Aggarwal, Bharat B.

AU - Al-Ubaidi, Muayyad R.

AU - Agarwal, Neeraj

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AB - We recently showed that photooxidative stress on cultured photoreceptor cells results in down-modulation of NF-κB activity which then leads to apoptosis of cultured 661W photoreceptor cells. In an effort to further delineate the mechanism of photoreceptor cell death, we sought to determine the effects of Bcl-2 overexpression on cell survivability. Wild-type 661W cells were transfected with the plasmid construct pSFFV-neo-Bcl-2 and several clones were isolated. All clones demonstrated increased Bcl-2 mRNA and protein levels, with the B4 clone exhibiting the greatest enhancement. On exposure to visible light the B4 cells were protected from undergoing apoptosis when compared with the mock transfected cells, as ascertained by TUNEL apoptosis assay and formazan based estimation of cell viability. The Bcl-2 overexpressing cells also maintained a higher Bcl-2/Bax ratio, suggesting that this ratio is important in protection from photooxidative stress. Electrophoretic mobility shift assays for NF-κB demonstrated higher activity in both nuclear and cytosolic fractions of the B4 photoreceptors compared with the 661W wild-type cells at all light exposure time points. Furthermore, the findings of the gel shift assays were further supported by immunocytochemistry for NF-κB which revealed that protein levels of the RelA subunit of NF-κB were protected in the nucleus as well as in the cytoplasm of Bcl-2 overexpressing B4 cells exposed to light compared to the 661W cells. These results suggest that Bcl-2 overexpression protects NF-κB protein levels and activity in the nucleus, indicating that preservation of NF-κB binding activity in the nucleus may be essential for photoreceptor cells to survive photooxidative damage induced apoptosis. copy& 2001 Academic Press.

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KW - Gene transfection

KW - NF-κB

KW - Photoreceptor cells

KW - Retinal dystrophies

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