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Pathways involved in proliferating, senescent and immortalized keratinocyte cell death mediated by two different
Jian-Zhong Qin1, Patricia E Bacon, Vijaya Chaturvedi
1Loyola University Medical Center, Skin Cancer Research Laboratory, Cardinal Bernardin Cancer Center, Maywood, IL 60153, USA. bnickol@lumc.edu
Abstract:
Properly regulated keratinocyte cell death is fundamentally important to maintain structural integrity and homeostatic function of epidermis. Moreover, from an oncological perspective, therapeutic approaches selectively targeting apoptosis of malignant cell types while sparing normal keratinocytes in surrounding skin is desirable. Apo2Ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) has been observed to preferentially induce cytopathic effects on transformed/malignant cell types compared with their non-neoplastic counterparts. In this report, two different biologically active preparations of Apo2L/TRAIL, a non-tagged version, NT-Apo2L/TRAIL, and a leucine zipper fusion protein, LZ-Apo2L/TRAIL, were examined for their ability to trigger apoptosis in normal human keratinocytes, and in an immortalized cell line (HaCaT cells). Differences between these preparations were observed, including: NT-Apo2L/TRAIL induced less keratinocyte apoptosis compared with LZ-Apo2L/TRAIL; NT-Apo2L/TRAIL also induced less apoptosis of HaCaT cells compared with LZ-Apo2L/TRAIL; LZ-Apo2L/TRAIL but not NT-Apo2L/TRAIL induced cytotoxic effects when keratinocytes became growth arrested due to undergoing spontaneous replicative senescence--a biological state previously observed to be resistant to UV-light-induced apoptosis. Similarities between preparations included: an enhanced ability for both Apo2L/TRAIL preparations to kill a greater relative percentage of HaCaT cells compared with keratinocytes; enhanced cytotoxicity towards keratinocytes that had their NF-B activity inhibited; a dependence of both Apo2L/TRAIL preparations on FADD and caspase activation; triggering of the same caspase cascades including caspase 8 and 3; and an ability to induce apoptosis even when HaCaT cells and keratinocytes were transduced to overexpress either Bcl-2 or Bcl-x(L) (survival factors that reduce susceptibility to UV-light-induced apoptosis). These results indicate that while both preparations of Apo2L/TRAIL possess biological activity, there are important differences as regards their ability to induce apoptosis in normal and immortalized keratinocytes. Moreover, the death receptor pathway triggered by LZ-Apo2L/TRAIL can overcome the apoptotic resistance normally observed in response to UV-light mediated by Bcl-2/Bcl-x(L), as well as by the state of cellular senescence. Unraveling the molecular basis for these differential biological effects may reveal a new strategic role for these death receptor/ligands linked to apoptosis in maintaining the dynamic balance of keratinocyte proliferation, differentiation, and cell death necessary to achieve a homeostatic thickness and function of normal skin. In addition, it may be possible to utilize these Apo2L/TRAIL preparations for the treatment of various sun-induced skin cancers as they can differentially trigger apoptosis of transformed keratinocytes, or keratinocytes with abnormal NF-kappaB signaling, while sparing adjacent normal keratinocytes.
Insights
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preparations differentially induce keratinocyte apoptosis. LZ-TRAIL shows greater efficacy, overcoming resistance in senescent cells and those overexpressing survival factors, suggesting therapeutic potential for skin cancers.
Area of Science:
- Cell Biology
- Molecular Biology
- Dermatology
Background:
- Keratinocyte cell death is crucial for epidermal homeostasis and cancer therapy.
- Apo2Ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) selectively targets malignant cells.
- Understanding TRAIL's differential effects on keratinocytes is key for therapeutic development.
Purpose of the Study:
- To compare the apoptosis-inducing abilities of two Apo2L/TRAIL preparations (NT-Apo2L/TRAIL and LZ-Apo2L/TRAIL) on normal and immortalized human keratinocytes.
- To investigate if Apo2L/TRAIL can overcome apoptotic resistance in senescent keratinocytes or those overexpressing survival factors (Bcl-2, Bcl-xL).
- To explore the potential of Apo2L/TRAIL in treating skin cancers by selectively targeting malignant keratinocytes.
Main Methods:
- Treatment of normal human keratinocytes and HaCaT cells with NT-Apo2L/TRAIL and LZ-Apo2L/TRAIL.
- Assessment of apoptosis induction, including in growth-arrested (senescent) keratinocytes and cells overexpressing Bcl-2 or Bcl-xL.
- Analysis of caspase activation pathways (caspase 8 and 3) and the role of FADD.
- Evaluation of effects on keratinocytes with inhibited NF-kappaB activity.
Main Results:
- LZ-Apo2L/TRAIL induced significantly more keratinocyte and HaCaT cell apoptosis than NT-Apo2L/TRAIL.
- LZ-Apo2L/TRAIL, but not NT-Apo2L/TRAIL, effectively induced apoptosis in senescent keratinocytes.
- Both TRAIL preparations triggered apoptosis dependent on FADD and caspase activation, utilizing caspase 8 and 3 cascades.
- Apoptotic resistance conferred by Bcl-2/Bcl-xL overexpression was overcome by LZ-Apo2L/TRAIL, particularly in senescent cells.
Conclusions:
- Both NT-Apo2L/TRAIL and LZ-Apo2L/TRAIL exhibit biological activity, but LZ-Apo2L/TRAIL demonstrates superior apoptosis-inducing capacity in keratinocytes.
- The LZ-Apo2L/TRAIL preparation can overcome apoptotic resistance mechanisms, including senescence and survival factor overexpression.
- These findings highlight the potential of LZ-Apo2L/TRAIL as a therapeutic agent for skin cancers, offering selective targeting of malignant keratinocytes while sparing normal cells.