p21
Cip1/WAF1
is the principle mediator of cell cycle arrest in response to DNA damage. p21 primarily mediates G
1
cell cycle arrest by inactivating G
1
-associated cyclin A- and cyclin E-containing ...cyclin/cdk complexes. In the present study we investigate the role of p21 in DNA damage-induced G
2
cell cycle arrest, particularly with respect to the G
2
-associated cyclin, cyclin B1. We demonstrate that cells lacking p21 or deficient in their ability to upregulate p21 are unable to mediate the downregulation of cyclin B1 in response to DNA damage as compared to wild-type cells. Decreased levels of cyclin B1 in response to DNA damage seen in wild-type cells is due to p21-mediated degradation of cyclin B1 as this can be inhibited by a proteasomal inhibitor. Cell cycle analysis reveals that p21-null cells are unable sustain G
2
cell cycle arrest and accumulate at greater than 4N DNA content. These results indicate that p21-mediated degradation of cyclin B1 in response to DNA damage is necessary for the maintenance of G
2
cell cycle arrest.
The protozoan
Leishmania chagasi expresses a surface metalloprotease, GP63, whose abundance increases 14-fold as parasites grow from logarithmic to stationary phase.
L. chagasi GP63 is encoded by ...three classes of
MSP genes that are differentially expressed during parasite growth. Using metabolic labeling and immunoprecipitation, we found
L. chagasi GP63 first appeared as a 66-kDa band that was replaced by a 63-kDa protein. This pattern also occurred in transfected
L. donovani harboring detectable products of only one
MSP gene, suggesting a precursor–product relationship. The half-life of GP63 increased from 29 h in logarithmic phase to >72 h in stationary phase promastigotes. GP63 loss from the cell was complemented by the appearance of a 63-kDa GP63 in extracellular medium in both membrane-associated and -free forms. Calculations suggested that the long and lengthening
T
1/2 of cell-associated GP63 accounts in part for its progressive accumulation in the cell during promastigote growth. The current findings add yet another level of complexity to post-transcriptionally regulated expression of an abundant surface molecule in a trypanosomatid protozoan.
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