Mechanism of ultraviolet B-induced cell cycle arrest in G2/M phase in immortalized skin keratinocytes with defective p53.
The ultraviolet B (UVB) portion (280-320 nm) of solar radiation is considered to be a major etiologic factor in human skin cancer and is a known cause of extensive DNA damage. In this study, we observed that UVB exposure of immortalized epidermal keratinocytes (HaCat cells) harboring mutant p53 leads to G(2)/M cell cycle arrest in both asynchronously growing and synchronized cells in a dose dependent manner. Following UVB exposure (200 mJ/cm(2)), we observed a threefold increase in G(2)/M population at 6 h, which increased to sixfold. The observed G(2)/M arrest was associated with an increase in cyclin B level whereas cdc2 protein remained unchanged. However, we observed an accumulation of tyrosine 15 hyperphosphorylated cyclin B-cdc2 complex. In addition, we observed an increase in chk1 kinase and a decrease in cdc25C protein levels. Chk1 phosphorylates cdc25C on serine 216 and inactivates it whereas cdc25C dephosphorylates tyrosine 15 phosphate of cdc2 and activates the cdc2-cyclin B complex. Therefore, the increase in chk1 and the decrease in cdc25C both participate in inhibiting the G2/M transition. Our data identifies two upstream targets leading to inhibition of cyclin B-cdc2 complexes, which explain the inhibition in cyclin B-associated cdc2 kinase following UVB exposure. The inactive phosphorylated cdc2-cyclin B complex remains sequestered in cytoplasm and may migrate to the nucleus following activation. Our data also indicate that UVB exerts unique effects in different types of skin keratinocytes having nonfunctional or mutant p53.
Athar
M
M
Department of Dermatology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, New York 10032, USA.
Kim
A L
AL
Ahmad
N
N
Mukhtar
H
H
Gautier
J
J
Bickers
D R
DR
D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle / Q000528:radiation effects*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002461:Cell Line, Transformed; D000071877:Checkpoint Kinase 1; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D005434:Flow Cytometry; D016195:G2 Phase / Q000528:radiation effects; D005786:Gene Expression Regulation / Q000528:radiation effects; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism / Q000528:radiation effects*; D008938:Mitosis / Q000528:radiation effects; D009154:Mutation / Q000235:genetics*; D010766:Phosphorylation / Q000528:radiation effects; D011494:Protein Kinases / Q000378:metabolism; D012867:Skin; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology*; D014466:Ultraviolet Rays; D020687:cdc25 Phosphatases / Q000378:metabolism
C534699:CCNB1 protein, human; D018797:Cell Cycle Proteins; D019926:Cyclin B; D056744:Cyclin B1; D016159:Tumor Suppressor Protein p53; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D016203:CDC2 Protein Kinase; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases