Tumor necrosis factor-alpha-associated mechanisms affecting the embryonic response to cyclophosphamide.
OBJECTIVE
We have previously shown that TNF-alpha(-/-) embryos are more sensitive to the exposure to cyclophosphamide (CP) compared with TNF-alpha(+/+) embryos; however, the underlying mechanisms are not fully understood. Thus, in our present study, we tried to identify those molecules that might be responsible for the protective effect of the cytokine.


METHODS
CP-treated TNF-alpha(-/-) and TNF-alpha(+/+) embryos were analyzed for changes in apoptosis by TUNEL and flow cytometry, while cell proliferation was analyzed by BrdU incorporation. The expression of Bax, bcl-2, p53, the p65 subunit of NF-kappaB and IkappaBalpha was assessed by Western blotting and immunohistochemistry.


RESULTS
CP-treated TNF-alpha(-/-) embryos exhibited a more profound decrease in their weight, which was accompanied by an earlier appearance of cellular damage and apoptotic cells and an earlier decrease in cell proliferation in the embryonic brain compared with TNF-alpha(+/+) embryos. Also, an increased percentage of Bax-positive cells and a decreased percentage of bcl-2-positive cells were detected in TNF-alpha(-/-) embryos 48 hr after exposure, which were accompanied by a decreased percentage of p53-positive cells.


CONCLUSIONS
Our data implicate TNF-alpha to be involved in the protection of the embryo against CP teratogenicity, possibly via alteration in Bax, bcl-2 or p53 expression.
Mammon
Keren
K
Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.
Savion
Shoshana
S
Orenstein
Hasida
H
Fein
Amos
A
Torchinsky
Arkady
A
Toder
Vladimir
V
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D003520:Cyclophosphamide / Q000494:pharmacology*; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D005260:Female; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; D003520:Cyclophosphamide