RLIP76 in defense of radiation poisoning.
OBJECTIVE
To determine the role of RLIP76 in providing protection from radiation and chemotherapy. In the present report, we used RLIP76 to refer to both the mouse (Ralbp1) and the human (RLIP76) 76-kDa splice variant proteins (RLIP76) for convenience and to avoid confusion. In other reports, Ralbp1 refers to the mouse enzyme (encoded by the Ralbp1 gene), which is structurally and functionally homologous to RLIP76, the human protein encoded by the human RALBP1 gene.


METHODS
Median lethal dose studies were performed in RLIP76(-/-) and RLIP76(+/+) C57B mice after treatment with a single dose of RLIP76 liposomes 14 h after whole body radiation. The radiosensitivity of the cultured mouse embryonic fibroblasts and the effects of buthionine sulfoximine (BSO), amifostine, c-jun N-terminal kinase (JNK), protein kinase B (Akt), and MAPK/ERK kinase (MEK) were determined by colony-forming assays. Glutathione-linked enzyme activities were measured by spectrophotometric assays, glutathione by dithiobis-2-nitrobenzoic acid (DTNB), lipid hydroperoxides by iodometric titration, and aldehydes and metabolites by thiobarbitauric acid reactive substances and liquid chromatography-mass spectrometry (LCMS).


RESULTS
RLIP76(-/-) mice were significantly more sensitive to radiation than were the wild-type, and RLIP76 liposomes prolonged survival in a dose-dependent manner in both genotypes. The levels of 4-hydroxynonenal and glutathione-conjugate of 4-hydroxynonenal were significantly increased in RLIP76(-/-) tissues compared with RLIP76(+/+). RLIP76(-/-) mouse embryonic fibroblasts were markedly more radiosensitive than RLIP76(+/+) mouse embryonic fibroblasts, despite increased glutathione levels in the former. RLIP76 augmentation had a remarkably greater protective effect compared with amifostine. The magnitude of effects of RLIP76 loss on radiation sensitivity was greater than those caused by perturbations of JNK, MEK, or Akt, and the effects of RLIP76 loss could not be completely compensated for by modulating the levels of these signaling proteins.


CONCLUSIONS
The results of our study have shown that RLIP76 plays a central role in radiation resistance.
Singhal
Jyotsana
J
Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Singhal
Sharad S
SS
Yadav
Sushma
S
Suzuki
Sumihiro
S
Warnke
Molly M
MM
Yacoub
Adly
A
Dent
Paul
P
Bae
Sejong
S
Sharma
Rajendra
R
Awasthi
Yogesh C
YC
Armstrong
Daniel W
DW
Awasthi
Sanjay
S
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D000447:Aldehydes / Q000378:metabolism; D004999:Amifostine / Q000494:pharmacology; D000818:Animals; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D005347:Fibroblasts / Q000528:radiation effects; D020690:GTPase-Activating Proteins / Q000172:deficiency / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008081:Liposomes; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011829:Radiation Dosage; D011836:Radiation Tolerance / Q000187:drug effects / Q000235:genetics; D011837:Radiation-Protective Agents / Q000494:pharmacology; D014410:Tumor Stem Cell Assay / Q000379:methods
D018528:ATP-Binding Cassette Transporters; D000447:Aldehydes; D020690:GTPase-Activating Proteins; D008081:Liposomes; C095637:RALBP1 protein, human; D011837:Radiation-Protective Agents; D019328:Buthionine Sulfoximine; D051057:Proto-Oncogene Proteins c-akt; D048031:JNK Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D005978:Glutathione; C027576:4-hydroxy-2-nonenal; D004999:Amifostine
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