Human cytomegalovirus (HCMV) stimulates arrested cells to enter the cell cycle by activating cyclindependent kinases (Cdks), notably Cdk2. Several mechanisms are involved in the activation of Cdk2. HCMV causes a substantial increase in the abundance of cyclin E and stimulates translocation of Cdk2 from the cytoplasm to the nucleus. Further, the abundance of the Cdk inhibitors (CKIs) p21 cip1/waf1 (p21 cip1 ) and p27 kip1 is substantially reduced. The activity of cyclin E/Cdk2 increases as levels of CKIs, particularly p21 cip1 , fall. We have previously shown that these phenomena contribute to priming the cell for efficient replication of HCMV. In this study, the mechanisms responsible for the decrease in p21 cip1 levels after HCMV infection were investigated by measuring p21 cip1 RNA and protein levels in permissive human lung (LU) fibroblasts after HCMV infection. Northern blot analysis revealed that p21 cip1 RNA levels increased briefly at 3 h after HCMV infection and then decreased to their nadir at 24 h; thereafter, RNA levels increased to about 60% of the preinfection level. Western blot analysis demonstrated that the relative abundance of p21 cip1 protein roughly paralleled the observed changes in initial RNA levels; however, the final levels of protein were much lower than preinfection levels. After a transient increase at 3 h postinfection, p21 cip1 abundance declined sharply over the next 24 h and remained at a very low level through 96 h postinfection. The disparity between p21 cip1 RNA and protein levels suggested that the degradation of p21 cip1 might be affected in HCMV-infected cells. Treatment of HCMV-infected cells with MG132, an inhibitor of proteasome-mediated proteolysis, provided substantial protection of p21 cip1 in mock-infected cells, but MG132 was much less effective in protecting p21 cip1 in HCMV-infected cells. The addition of E64d or Z-Leu-Leu-H, each an inhibitor of calpain activity, to HCMVinfected cells substantially increased the abundance of p21 cip1 in a concentration-dependent manner. To verify that p21 cip1 was a substrate for calpain, purified recombinant p21 cip1 was incubated with either m-calpain or -calpain, which resulted in rapid proteolysis of p21 cip1 . E64d inhibited the proteolysis of p21 cip1 catalyzed by either m-calpain or -calpain. Direct measurement of calpain activity in HCMV-infected LU cells indicated that HCMV infection induced a substantial and sustained increase in calpain activity, although there was no change in the abundance of either m-or -calpain or the endogenous calpain inhibitor calpastatin. The observed increase of calpain activity was consistent with the increases in intracellular free Ca 2؉ and phospholipid degradation in HCMV-infected LU cells reported previously from our laboratory. Considered together, these results suggest that the increase in calpain activity observed following HCMV infection contributes significantly to the reduction of p21 cip1 levels and the resultant cell cycle progression.Human cytomegalovirus (HCMV) infection is wides...
Human cytomegalovirus (HCMV) induces serum-or densityarrested human lung (LU) cells to traverse the cell cycle, providing it with a strategy to replicate in post-mitotic cells that are its cellular substrate in vivo. HCMV infection also induces high cellular levels of p53, seemingly in contradiction to the observed cell cycle progression. This study was undertaken to examine the mechanism(s) of the increased p53 abundance. HCMV infection caused a 4-fold increase in p53 that preceded a substantial increase in p53 transcripts by more than 24 h. p53 was stabilized in HCMV-infected cells (from a half-life of less than 30 min to about 8 h) and was less sensitive to proteasome-mediated degradation. Ubiquitination of p53 in mock-infected LU cells was sensitive to inhibition by trans-4-iodo, 4 -boranylchalcone, consistent with HDM2-catalyzing ubiquitination of p53. In HCMV-infected cells, ubiquitination of p53 was essentially undetectable. Although HDM2 had a nuclear distribution in mock-infected LU cells, in HCMV-infected cells HDM2 was translocated to the cytoplasm beginning at 12 h and demonstrated decreased cellular abundance thereafter. HDM2 was stabilized in the HCMV-infected cells by MG132, indicating a shift from p53 to HDM2 ubiquitination. p53 demonstrated a predominantly nuclear distribution in HCMV-infected cells through 48 h, resulting in p53 and HDM2 in distinct subcellular compartments. The principal mechanism responsible for increased p53 stabilization was nuclear export and degradation of HDM2. Thus, HCMV uses a shift from p53 to HDM2 ubiquitination and destabilization to obtain protracted high levels of p53, while promoting cell cycle traverse.Human cytomegalovirus (HCMV), 2 a large -herpesvirus, infects the majority of humans, producing life long persistent infection (1, 2). Efficient replication of HCMV in stationary cells is dependent on the progression of infected cells through the cell cycle to a point at or near the G 1 /S boundary (3, 4). Consistent with this requirement, HCMV infection stimulates potent mitogenic signaling in infected cells, with some evidence suggesting that HCMV mitogenic signaling is more robust than that of serum growth factors, because HCMV, but not serum growth factors, induces the entry of density-arrested cells into the cell cycle. Among the immediate early and early signaling events induced by HCMV are hydrolysis of phosphatidylinositol 4,5-bisphosphate (5), activation of phospholipases (6), increased cellular levels of secondary messengers (3, 6, 7), production of robust and protracted signaling from some components of these pathways (8), stimulation of several protein kinases (6, 9), and activation of a number of cellular transcription factors (9, 10). Some of this signaling derives from HCMV binding to its cellular receptors (epidermal growth factor receptor (11) and integrin ␣v3 (12)), but HCMV infection also modifies the cell with induction of cellular (e.g. activation of m-and -calpains (13)) and viral (e.g. cellular transcriptional activation by IE2-86 (14)) ac...
RLIP76 (RALBP1) is a multifunctional transporter involved in signaling and transmembrane movement of solute allocrites, which include glutathione conjugates and several natural product antineoplastic agents [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334; (2001) Biochemistry 40, 4159-4168]. Our previous studies suggested that the membrane-anchoring domain resides in the N-terminus of RLIP76, despite the lack of identifiable membrane-spanning domains. Amino acid sequence analysis indicated that this region of RLIP76 contains sequences that are similar to those of vector peptides. We, therefore, have studied the effect of a series of deletion mutant proteins on hydrophobicity and transport activity. RLIP76 or one of its derived deletion mutants was expressed in Escherichia coli, and bacteria were lysed and extracted in buffer without or with the nonionic detergent polidocanol. The ratio of RLIP76 in the detergent/aqueous extracts was found to be 2.5 for the wild-type protein, but decreased to 0.7 in the mutant in which amino acids 154-219 were deleted. Deletion of only one segment of this region (amino acids 171-185) alone resulted in a significant decrease in this ratio to 1.0. For the mutants with deletions within the region from amino acid 154 to 219, loss of hydrophobicity correlated with less incorporation of mutants into artificial liposomes, and decreased transport activity toward doxorubicin and dinitrophenyl-S-glutathione. In contrast, deletion of one of the two ATP-binding sites (at amino acids 65-80 or 415-448) or both sites did not affect hydrophobicity but reduced or abrogated transport activity. NSCLC (H358) stably transfected with del171-185 and del154-219 showed that loss of these regions results in a decrease in the extent of membrane association of RLIP76. Confocal laser immunohistochemistry colocalized amino acids 171-185 with her2/neu on the cell surface. Depletion of wild-type RLIP76 using si-RNA directed to this region in cells transfected with del171-185 resulted in the loss of cell surface expression. These finding demonstrate that amino acids 171-185 constitute a cell surface epitope which is necessary for optimal transport of anthracycline and glutathione conjugates by RLIP76, and that this peptide could be a novel target for antineoplastic therapy.
1,3-Butadiene (BD) causes genetic damage, including adduct formation, sister chomatid exchange, and point mutations. Previous studies have focused on the types of genetic damage and tumors found after long-term exposure of rodents to butadiene. This study examined the effect of the most active BD metabolite, butadiene diepoxide (BDO2), on cell cycle entry and progression in human lung fibroblasts (LU cells) with a normal diploid karyotype. Serum-arrested (G0) LU cells were exposed to BDO2 for 1 hr and stimulated to divide with medium containing 10% fetal bovine serum. The BDO2-treated LU cells were evaluated for cell cycle progression, nuclear localization of arrest mediators, mitotic index, and cellular proliferation. The BDO2-treated cells demonstrated a substantial inhibition of cell proliferation when treated with 100 microM BDO2 for 1 hr. No appreciable levels of apoptosis or mitotic figures were observed in the BDO2-treated cells through 96 hr posttreatment. Flow cytometric analysis revealed that the lack of proliferation in BDO2-treated LU cells was related to G1 arrest in about half of the cells and a delayed progression through S and G2 arrest in nearly all of the remaining cells. Both G1 and G2 arrest were prolonged and only a very small percentage of BDO2-treated cells were eventually able to replicate. Increased nuclear localization of both p53 and p21(cip1) was observed in BDO2-treated cells, suggesting that the cell cycle arrest was p21(cip1)-mediated. These results demonstrate that BDO2 induces cell cycle perturbation and arrest even with short-term exposure that does not produce other pathologic cellular effects.
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