Secalonic acid D blocks embryonic palatal mesenchymal cell-cycle by altering the activity of CDK2 and the expression of p21 and cyclin E.
BACKGROUND
The mycotoxin, secalonic acid D (SAD), a known animal and potential human cleft palate (CP)-inducing agent, is produced by Pencillium oxalicum in corn. SAD selectively inhibits proliferation of murine embryonic palatal mesenchymal (MEPM) cells leading to a reduction in cell numbers. These effects can explain the reduction in shelf size and the resulting CP seen in the offspring of SAD-exposed mice. Ability of SAD to inhibit proliferation as well as to block the progression of cells from G1- to S-phase of the cell-cycle were also shown in the human embryonic palatal mesenchymal (HEPM) cells suggesting the potential CP-inducing effect of SAD in human beings


METHODS
Gestation day (GD) 12 mouse embryos and HEPM cells were used to test the hypothesis that the cell-cycle block induced by SAD results from a disruption of stage-specific regulatory components both in vivo and in vitro. The effects of SAD on the activity of various cyclin dependent kinases (CDK) and on the levels of various positive (cyclins and CDK) and negative (CDK inhibitors p15, 16, 18, 19, 21, 27, 57) cell-cycle regulators were assessed by performing kinase assays and immunoblots, respectively.


RESULTS
In the murine embryonic palates, SAD specifically inhibited G1/S-phase-specific CDK2 activity, reduced the level of cyclin E and tended to increase the level of the CIP/kip CDK inhibitor, p21. In the HEPM cell cultures, exposure to IC50 of SAD significantly affected all of the above targets. In addition, a reduction in the levels/activity of CDK 4/6, a reduction in the levels of cyclins D1, D2, D3, E, A, and all INK4 family proteins, and an increase in the level of the CIP/kip CDK inhibitor, p57, were also seen.


CONCLUSIONS
These results suggest that the S-phase-specific cell-cycle proteins CDK2, cyclin E and possibly p21 are the common targets of SAD in murine palatal shelves in vivo and in human embryonic palatal mesenchymal cells in vitro and may be relevant to the pathogenesis of SAD-induced CP.
Dhulipala
V C
VC
Department of Biomedical Sciences, University of Missouri, Columbia, Missouri 65211, USA.
Maddali
K K
KK
Welshons
W V
WV
Reddy
C S
CS
D000818:Animals; D042846:CDC2-CDC28 Kinases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002972:Cleft Palate / Q000139:chemically induced*; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D047108:Embryonic Development / Q000187:drug effects; D006801:Humans; D008648:Mesoderm / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D010159:Palate / Q000166:cytology / Q000187:drug effects* / Q000196:embryology; D044004:Xanthones / Q000633:toxicity*
C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D018797:Cell Cycle Proteins; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D044004:Xanthones; C009218:secalonic acid; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2