Figure 1Generation of epithelial cells with altered NF-κB function. Normal keratinocytes were transduced with retroviral expression vector for (a) lacZ normal control, (b) constitutively active p50, and (c) the trans-dominant IκBαM super-repressor, then subjected to immunofluorescence staining with antibody to p50 (bars = 5 µm). Note marked nuclear expression in p50-transduced cells and blockade of nuclear-localized p50 in IκBαM.NF-κB subunit transcription activation domains in that these changes are not observed with transcriptionally inactive mutants; the basis for this effect is unclear. This approach, however, has been shown previously to produce the predicted respective induction or blockade of NF-κB-driven gene expression in epithelial cells, both in the case of p50 and p65 alone as well as both subunits together (21) and provides a basis for determining the effects altering NF-κB function on apoptosis in this setting.Fas is expressed in epithelial cells and is implicated as a potentially important mediator of epithelial cell death in settings of inflammation (33-35) and ultraviolet injury (5,36). Cotransduction with vectors altering NF-κB function and a retroviral vector for human Fas/CD95 was used to study NF-κB effects on Fas-triggered apoptosis in epithelial cells. Fas activation in normal control and in IκBαM[+] cells leads to rapid cellular rounding, shrinkage, and detachment in the majority of cells; however, these changes are not seen in NF-κB subunit-expressing cells (Figure 2, a-c and f). These morphologic changes are consistent with apoptosis, a possibility supported by detection of DNA strand breakage using TUNEL assay and characteristic nuclear morphologic condensation and collapse in these cells (Figure 2, d and e).TNFα is another known trigger of apoptosis in many tissues that also impacts NF-κB function. TNFα activation of NF-κB through the TNF receptor TNFR1 opposes TNFα-induced apoptosis in a number of cell types in a process dependent on TRAF2 (8, 37). Whereas TNFα failed to alter normal keratinocytes or those expressing active NF-κB subunits, NF-κB blockade renders these cells very susceptible to TNFα-induced apoptosis (Figure 3, a-d), suggesting an analogous role for NF-κB in preventing apoptosis in epithelial cells.Blockade of NF-κB function leads to premature epidermal cell apoptosis in vivo. Normal stratified epithelium maintains a balance between cellular proliferation and a specialized form of programmed cell death confined to the outer differentiated cell layer at the stratum granulosum-stratum corneum interface (4). This terminal differentiation-associated cell death is not accompanied by classic cell morphologic features of apoptosis such as cell shrinkage and collapse, membrane blebbing, and nuclear condensation seen in other cell death settings in epithelium such as infection and ultraviolet injury (38). NF-κB translocates into nuclei of cells within outer layers of stratified epithelium (21), and this translocation is accompanied by NF-κB target gene activation (K. Hinata et...

We investigated the role of the cyclin-dependent kinase inhibitors p21Cip1 and p27 Kip1 in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bromodeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DNA replication is rapidly shut down in immortalized mouse embryo fibroblasts. This S-phase arrest is intact in fibroblasts lacking the cyclin kinase inhibitors p21Cip1 and p27 Kip1 , indicating that these molecules are not essential elements of the arrest pathway. Hypoxia-induced arrest is accompanied by dephosphorylation of pRb and inhibition of cyclindependent kinase 2, which results in part from inhibitory phosphorylation. Interestingly, cells lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this response. Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerobic levels by 6 h. Cells lacking p21, however, resume DNA synthesis more rapidly upon reoxygenation than wild-type cells, suggesting that this inhibitor may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phase even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored more rapidly in the doubleknockout cells when returned to normoxia. These studies reveal that restoration of DNA synthesis after hypoxic stress, but not the S phase arrest itself, is regulated by p21 and p27.

Due to the abnormal vasculature of solid tumors, tumor cell oxygenation can change rapidly with the opening and closing of blood vessels, leading to the activation of both hypoxic response pathways and oxidative stress pathways upon reoxygenation. Here, we report that ataxia telangiectasia mutated-dependent phosphorylation and activation of Chk2 occur in the absence of DNA damage during hypoxia and are maintained during reoxygenation in response to DNA damage. Our studies involving oxidative damage show that Chk2 is required for G 2 arrest. Following exposure to both hypoxia and reoxygenation, Chk2 ؊/؊ cells exhibit an attenuated G 2 arrest, increased apoptosis, reduced clonogenic survival, and deficient phosphorylation of downstream targets. These studies indicate that the combination of hypoxia and reoxygenation results in a G 2 checkpoint response that is dependent on the tumor suppressor Chk2 and that this checkpoint response is essential for tumor cell adaptation to changes that result from the cycling nature of hypoxia and reoxygenation found in solid tumors.

Although shown to be highly expressed by the epidermis in inflammatory skin disease, the ability of the Fas protein to trigger apoptosis in the distinct cell subpopulations of cutaneous tissue, particularly with regard to receptor density and the degree of crosslinking, has not been fully characterized. We therefore determined the effect of Fas cross-linking in primary human dermal fibroblasts at both high and low levels of Fas receptor expression. First, we examined the effects of the anti-Fas monoclonal antibody, CH-11, on fibroblasts expressing low basal levels of Fas. In these cells Fas aggregation stimulated proliferation by 160 +/- 10% over untreated controls. In contrast, the same concentration of CH-11 had an inhibitory effect on epidermal keratinocyte growth. Because Fas is upregulated in inflamed skin, we next examined the effects of Fas cross-linking on fibroblasts expressing augmented levels ofFas. Fibroblasts were either transfected with plasmids for overexpression of full length or bioengineered Fas receptors or were transduced with a retroviral Fas expression vector. In these cells Fas oligomerization triggered the morphologic changes indicative of apoptosis regardless of whether or not the Fas-signaling domain was tethered to the plasma membrane. These studies indicate that Fas oligomerization in dermal fibroblasts may initiate dual signaling programs, either proliferation or apoptosis, and that the chosen outcome may depend upon the magnitude of Fas aggregation.