Shedding LIGHT (TNFSF14) on the tumor microenvironment of colorectal cancer liver metastases.

BACKGROUND: T-cell infiltration in primary colon tumors is associated with improved patient survival. Preliminary data supports a similar association in colorectal liver metastases (CRLM), and we previously identified increased CRLM expression of the immunostimulatory cytokine LIGHT (TNFSF14) to be related to improved patient prognosis. Therefore, mechanisms to augment the T-cell response in CRLM may be a promising treatment modality, however, the tumor immune microenvironment and LIGHT expression in CRLM remains to be characterized. METHODS: Utilizing a syngeneic and immunocompetent model of CRLM, the immune microenvironment was characterized for lymphocyte phenotype, function, and location utilizing flow cytometry, immunoassays, and immunofluorescence microscopy. RESULTS: CD3+ and CD4+ lymphocytes were decreased, and CD8+ cells were increased in CRLM compared to control liver. When present, greater populations of tumor infiltrating lymphocytes (TIL) were found peritumoral than intratumoral. The TIL expressed significantly higher levels of CD69 and CD107a, but lower levels of LIGHT. Cytokine expression profiles revealed increased levels of the T-helper 1 (Th1) cytokines IFN gamma, IL-12, IL-1b, and IL-8 in CRLM compared to control liver tissue. There was no difference in T-helper 2 (Th2) cytokines between the groups. CONCLUSIONS: Characterization of the tumor microenvironment of CRLM revealed that although a limited number of activated T-cells infiltrate the tumor and initiate an immune response, the number of LIGHT + T cells infiltrating the tumor were very low. Techniques to decrease suppressive influences or augment the cytotoxic T-cell response are needed and may be possible through mechanisms that can increase intratumoral TIL LIGHT expression.

Cutting edge: A critical functional role for IL-23 in psoriasis.

Interleukin-23 is a key cytokine involved in the generation of Th17 effector cells. Clinical efficacy of an anti-p40 mAb blocking both IL-12 and IL-23 and disease association with single nucleotide polymorphisms in the IL23R gene raise the question of a functional role of IL-23 in psoriasis. In this study, we provide a comprehensive analysis of IL-23 and its receptor in psoriasis and demonstrate its functional importance in a disease-relevant model system. The expression of IL-23 and its receptor was increased in the tissues of patients with psoriasis. Injection of a mAb specifically neutralizing human IL-23 showed IL-23-dependent inhibition of psoriasis development comparable to the use of anti-TNF blockers in a clinically relevant xenotransplant mouse model of psoriasis. Together, our results identify a critical functional role for IL-23 in psoriasis and provide the rationale for new treatment strategies in chronic epithelial inflammatory disorders.

Fingertip reconstruction with a flap based on the dorsal branch of the digital artery at the middle phalanx: a simple and reliable flap.

BACKGROUND: It is of utmost importance for hand surgeons to rehabilitate injured fingertips aesthetically and functionally. The purpose of our study was to investigate the effect of the dorsal branch (DB) finger flap on fingertip reconstruction. METHOD: We used the flap based on the DB of the proper digital artery for fingertip reconstruction in 3 patients at the primary stage. The size of the flaps ranged from 1.5 × 2.0 cm to 2.5 × 4.5 cm. RESULT: The DB finger flaps in our series survived uneventfully during the follow-up period (range, 4-6 months). All the patients were contented with the aesthetic and functional outcomes of the flaps. The average static and moving 2-point discrimination of the flaps was 8.5 mm and 7.2 mm, respectively. CONCLUSION: This sort of flap based on the DB of the proper digital artery was a simple and reliable alternative to reconstruct fingertip defects at the primary stage.

Isoform-specific and cell/tissue-dependent effects of p38 MAPKs in regulating inflammation and inflammation-associated oncogenesis.

p38 MAPK (mitogen-activated protein kinases) family proteins (α, ß, γ and δ) are key inflammatory kinases and play an important role in relaying and processing intrinsic and extrinsic signals in response to inflammation, stress, and oncogene to regulate cell growth, cell death and cell transformation. Recent studies in genetic mouse models revealed that p38α in epithelial cells mostly suppresses whereas in immune cells it promotes inflammation and inflammation-associated oncogenesis. On the contrary, p38γ and p38δ signaling in immune and epithelial cells is both pro-inflammatory and oncogenic. This review summarizes recent discoveries in this field, discusses possible associated mechanisms, and highlights potentials of systemically targeting isoform-specific p38 MAPKs. Understanding of p38 MAPK isoform-specific and cell/tissue- and perhaps stage-dependent effects and their integrated regulated activity in inflammation and in inflammation-associated oncogenesis is essential for effectively targeting this group of kinases for therapeutic intervention.

Implantation of nanofibrous silk scaffolds seeded with bone marrow stromal cells promotes spinal cord regeneration (6686 words).

Spinal cord injury (SCI) is a common pathology often resulting in permanent loss of sensory, motor, and autonomic function. Numerous studies in which stem cells have been transplanted in biomaterial scaffolds into animals have demonstrated their considerable potential for recovery from SCI. In the present study, a three-dimensional porous silk fibroin (SF) scaffold with a mean pore size of approximately 383 µm and nanofibrous structure was fabricated, the silk scaffold enabling the enhanced attachment and proliferation of bone marrow stromal cells (BMSCs). Investigation of its therapeutic potential was conducted by implantation of the nanofibrous SF scaffold seeded with BMSCs into a transected spinal cord model. Recovery of the damaged spinal cord was significantly improved after 2 months, compared with a non-nanofibrous scaffold, in combination with decreased glial fibrillary acidic protein (GFAP) expression and improved axonal regeneration at the site of injury. Furthermore, elevated Basso-Beattie-Bresnahan (BBB) scores indicated greatly improved hindlimb movement. Together, these results demonstrate that transplantation of neural scaffolds consisting of nanofibrous SF and BMSCs is an attractive strategy for the promotion of functional recovery following SCI.

Consequences of p16 tumor suppressor gene inactivation in mycosis fungoides and Sézary syndrome and role of the bmi-1 and ras oncogenes in disease progression.

In examining the expression of oncogenes and tumor suppressor genes in mycosis fungoides and Sézary syndrome, we found the cell cycle-regulating protein p16 to be absent in T cells. Immunohistochemical staining with p16-specific antibodies showed that the number of p16-expressing cells in cutaneous lesions decreases in late stages. The repression of p16 was not attributable to deletion or methylation of this gene; however, the Bmi-1 oncogene, a known suppressor of p16, was present in mycosis fungoides and Sézary syndrome cell lines and skin lesions. The absence of p16 correlated with the phosphorylation of the retinoblastoma protein on cyclin D/CDK4- or cyclin D/CDK6-specific sites. Ki-ras, which stimulates phosphorylation of retinoblastoma via cyclin-dependent kinases, was found in all tested cutaneous T-cell lymphoma samples; and its expression generally was stronger in advanced stages. Thus, cutaneous T-cell lymphoma cells show changes in oncogene and tumor suppressor gene expression that increase proliferation.

Immunopathogenesis of psoriasis.

Investigations into the cause and treatment of psoriasis remain at the forefront of basic and applied clinical research efforts around the world. The purpose for this review is to provide an up-to-date synopsis of recent progress in ten sections exploring the immunological and inflammatory basis for psoriasis. Given the breadth of this topic in investigative skin biology and frequent paradigm shifts, it should not be surprising that the bibliography contains more than 150 references; many of which have been published in the last 5 years. Whereas considerable progress has been made into the immunopathogenesis of psoriasis, many fundamentally important questions remain regarding the role of cells located in both epidermal and dermal compartments. Attempts to characterize various animal models of psoriasis, delineation of the mechanism of action for biological agents, and consideration of molecular links between skin inflammation and various extracutaneous comorbidities are likely to continue challenging investigators and clinicians for many years to come.

The cytokine and chemokine network in psoriasis.

Creation and maintenance of psoriatic plaques require a multicellular conspiracy by which prepsoriatic skin becomes infiltrated by a variety of immunocytes triggering changes in the behavior of epidermal keratinocytes and endothelial cells. These complex cellular events require coordination in space and time to achieve the mature plaque. Key molecular coordinators dictating behavior and movement of cells within plaques include cytokines as well as chemokines. These mediators of inflammation play fundamentally important roles in the pathophysiology of psoriasis. The purpose of this chapter is to provide an updated review of cytokine and chemokine networks in psoriatic skin lesions.

Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells.

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.

Knockdown of p53 levels in human keratinocytes accelerates Mcl-1 and Bcl-x(L) reduction thereby enhancing UV-light induced apoptosis.

Ultraviolet (UV) light exposure is a common cause of epithelial-derived skin cancers, and the epidermal response to UV-light has been extensively studied using both mouse models and cultured human keratinocytes (KCs). Elimination of cells with UV-induced DNA damage via apoptosis provides a powerful mechanism to minimize retention or expansion of genetically abnormal cells. This cell editing function has largely been ascribed to the biological role of the p53 tumor suppressor gene, as mutations or deletions involving p53 have been linked to skin cancer development. Rather than introducing mutations, or using cells with complete loss of wild-type p53, we used an siRNA-based approach to knockdown, but not eliminate, p53 levels in primary cultures of human KCs followed by UV-irradiation. Surprisingly, when p53 levels were reduced by 50-80% the apoptosis induced by exposure to UV-light was accelerated and markedly enhanced (two- to three- fold) compared to control siRNA treated KCs. The p53 siRNA treated KCs were characterized by elevated E2F-1 levels accompanied by accelerated elimination of the Mcl-1 and Bcl-x(L) antiapoptotic proteins, as well as enhanced Bax oligomerization. Forced overexpression of either Mcl-1 or Bcl-x(L) reduced the UV-light enhanced apoptotic response in p53 siRNA treated KCs. We conclude that p53 not only can provide proapoptotic signals but also regulates a survival pathway influencing Mcl-1 and Bcl-x(L) levels. This overlooked survival function of p53 may explain previous paradoxical responses noted by investigators using p53 heterozygous and knockout mouse models, and opens up the possibility that not all liaisons within the cell involving p53 necessarily represent fatal attractions.

Knock down of p53 levels in human keratinocytes increases susceptibility to type I and type II interferon-induced apoptosis mediated by a TRAIL dependent pathway.

BACKGROUND: Keratinocytes (KCs) in healthy skin only undergo death following differentiation to produce stratum corneum. By contrast, in inflammatory pathological conditions featuring type I (IFN-alpha) and type II (IFN-gamma) interferons KCs undergo premature apoptosis. OBJECTIVE: To define apoptotic susceptibility of KCs, response to interferons was examined. Since molecular cross-talk occurs between interferons and p53, potential mechanistic roles for p53 in KC apoptosis were investigated. METHODS: Knock down of p53 was performed, and apoptotic response to addition of interferons was assessed using FACS and by staining for activated caspase 3 and TUNEL. Elucidation of death pathway was accomplished by using a dominant negative death receptor construct and a neutralizing TRAIL antibody. RESULTS: Reduction in p53 levels in KCs by siRNA treatment enhanced, rather than reduced, apoptotic responses to IFN-alpha plus IFN-gamma. In an immortalized human KC cell line (HaCaT cells with both p53 alleles mutated) enhanced apoptotic susceptibility to interferon exposure was also observed. The mechanism for this enhanced apoptosis involved induction of TRAIL and its interaction with death receptors, as blocking the death receptor pathway using dominant negative FADD, or by addition of neutralizing antibody against TRAIL, reduced the apoptotic response to IFN-alpha and IFN-gamma. CONCLUSION: These results indicate IFN-alpha plus IFN-gamma triggers apoptosis independent of p53 in HaCaT cells, and also demonstrate an unexpected survival role for p53 in human KCs as regards apoptotic responsiveness to cytokines such as IFN-alpha and IFN-gamma involving activation of TRAIL-related death receptors. Strategies enhancing p53 regulated survival proteins in KCs may be of therapeutic benefit in skin disorders characterized by activated immunocytes triggering premature KC apoptosis.

Regulation of apoptosis by p53 in UV-irradiated human epidermis, psoriatic plaques and senescent keratinocytes.

The carcinogenic effects of sunlight in human epidermis may be thwarted by either: transient growth arrest and repair of DNA photodamage in keratinocytes (KCs); elimination of KCs with damaged DNA via apoptosis; or by stimulating a senescence switch whereby KCs become irreversibly growth arrested. Using normal human skin organ cultures and living epidermal equivalents, we demonstrate that in the proliferative basal layer, removal of KCs via apoptosis had a rapid onset (beginning within 2 h) following UV-light exposure generating progressively greater numbers of KCs with thymine dimers as the dose of UV-light was increased; involved induction of Apaf-1, activation of caspase-3, and was dependent on p53 activation as addition of a p53 chemical inhibitor blocked the apoptotic response. Suprabasal layer KCs underwent apoptosis at much later time points (>8 h). KCs in the basal layer repaired DNA damage more rapidly than KCs in suprabasal layers. Steady state levels of p53 increased in irradiated cells, and the increase was accompanied by phosphorylation of serine 9 and serine 15, but not serine 6 residues. By contrast, cultured KCs undergoing spontaneous replicative senescence were resistant to UV-induced apoptosis. Senescent KCs constitutively contained low levels of p53, which were neither increased nor phosphorylated or acetylated after UV-exposure and possessed minimal DNA binding activity, indicative of functional inactivation. Furthermore, treatment of senescent KCs with DNA damaging agent adriamycin did not result in activation of latent p53 or apoptosis. When KCs within psoriatic plaques were examined, they resembled senescent KCs in that they expressed p53, which was not phosphorylated or acetylated. Thus, UV-light induces DNA damage in human epidermal KCs triggering p53 activation, and subsequent apoptosis involving distinct cell layers and kinetics. However, the lack of p53 activation as seen in senescent KCs and psoriatic plaques, is associated with a relative resistance of KCs to UV-induced apoptosis. In conclusion, the sensitivity and resistance of KCs to apoptosis depends not only on the location within various layers of epidermis and levels of p53, but may also involve p53 activation via post-translational modifications.

p53-independent NOXA induction overcomes apoptotic resistance of malignant melanomas.

Once melanoma metastasizes, no effective treatment modalities prolong survival in most patients. This notorious refractoriness to therapy challenges investigators to identify agents that overcome melanoma resistance to apoptosis. Whereas many survival pathways contribute to the death-defying phenotype in melanoma, a defect in apoptotic machinery previously highlighted inactivation of Apaf-1, an apoptosome component engaged after mitochondrial damage. During studies involving Notch signaling in melanoma, we observed a gamma-secretase tripeptide inhibitor (GSI; z-Leu-Leu-Nle-CHO), selected from a group of compounds originally used in Alzheimer's disease, induced apoptosis in nine of nine melanoma lines. GSI only induced G2-M growth arrest (but not killing) in five of five normal melanocyte cultures tested. Effective killing of melanoma cells by GSI involved new protein synthesis and a mitochondrial-based pathway mediated by up-regulation of BH3-only members (Bim and NOXA). p53 activation was not necessary for up-regulation of NOXA in melanoma cells. Blocking GSI-induced NOXA using an antisense (but not control) oligonucleotide significantly reduced the apoptotic response. GSI also killed melanoma cell lines with low Apaf-1 levels. We conclude that GSI is highly effective in killing melanoma cells while sparing normal melanocytes. Direct enhancement of BH3-only proteins executes an apoptotic program overcoming resistance of this lethal tumor. Identification of a p53-independent apoptotic pathway in melanoma cells, including cells with low Apaf-1, bypasses an impediment to current cytotoxic therapy and provides new targets for future therapeutic trials involving chemoresistant tumors.

Skin immune sentinels in health and disease.

Human skin and its immune cells provide essential protection of the human body from injury and infection. Recent studies reinforce the importance of keratinocytes as sensors of danger through alert systems such as the inflammasome. In addition, newly identified CD103(+) dendritic cells are strategically positioned for cross-presentation of skin-tropic pathogens and accumulating data highlight a key role of tissue-resident rather than circulating T cells in skin homeostasis and pathology. This Review focuses on recent progress in dissecting the functional role of skin immune cells in skin disease.

Cross-talk between notch and the estrogen receptor in breast cancer suggests novel therapeutic approaches.

High expression of Notch-1 and Jagged-1 mRNA correlates with poor prognosis in breast cancer. Elucidating the cross-talk between Notch and other major breast cancer pathways is necessary to determine which patients may benefit from Notch inhibitors, which agents should be combined with them, and which biomarkers indicate Notch activity in vivo. We explored expression of Notch receptors and ligands in clinical specimens, as well as activity, regulation, and effectors of Notch signaling using cell lines and xenografts. Ductal and lobular carcinomas commonly expressed Notch-1, Notch-4, and Jagged-1 at variable levels. However, in breast cancer cell lines, Notch-induced transcriptional activity did not correlate with Notch receptor levels and was highest in estrogen receptor alpha-negative (ERalpha(-)), Her2/Neu nonoverexpressing cells. In ERalpha(+) cells, estradiol inhibited Notch activity and Notch-1(IC) nuclear levels and affected Notch-1 cellular distribution. Tamoxifen and raloxifene blocked this effect, reactivating Notch. Notch-1 induced Notch-4. Notch-4 expression in clinical specimens correlated with proliferation (Ki67). In MDA-MB231 (ERalpha(-)) cells, Notch-1 knockdown or gamma-secretase inhibition decreased cyclins A and B1, causing G(2) arrest, p53-independent induction of NOXA, and death. In T47D:A18 (ERalpha(+)) cells, the same targets were affected, and Notch inhibition potentiated the effects of tamoxifen. In vivo, gamma-secretase inhibitor treatment arrested the growth of MDA-MB231 tumors and, in combination with tamoxifen, caused regression of T47D:A18 tumors. Our data indicate that combinations of antiestrogens and Notch inhibitors may be effective in ERalpha(+) breast cancers and that Notch signaling is a potential therapeutic target in ERalpha(-) breast cancers.

p38alpha antagonizes p38gamma activity through c-Jun-dependent ubiquitin-proteasome pathways in regulating Ras transformation and stress response.

p38 MAPK family consists of four isoform proteins (alpha, beta, gamma, and delta) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38alpha may regulate Ras transformation and stress response through depleting its family member p38gamma protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38alpha (MKK6-p38alpha) and p38gamma (MKK6-p38gamma) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38alpha phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38gamma protein expression. p38gamma, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38alpha phosphorylation decreases p38gamma protein expression, whereas its inhibition increases cellular p38gamma concentrations, indicating an active role of p38alpha phosphorylation in negatively regulating p38gamma protein expression. Mechanistic analyses show that p38alpha requires c-Jun activation to deplete p38gamma proteins by ubiquitin-proteasome pathways. These results suggest that p38alpha may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38gamma family protein.

Enhanced killing of melanoma cells by simultaneously targeting Mcl-1 and NOXA.

By deciphering the dysregulation of apoptosis in melanoma cells, new treatment approaches exploiting aberrant control mechanisms regulating cell death can be envisioned. Among the Bcl-2 family, a BH3-only member, NOXA, functions in a specific mitochondrial-based cell death pathway when melanoma cells are exposed to a proteasome inhibitor (e.g., bortezomib). Some therapeutic agents, such as bortezomib, not only induce proapoptotic Bcl-2 family members and active conformational changes in Bak and Bax but also are associated with undesirable effects, including accumulation of antiapoptotic proteins, such as Mcl-1. To enhance the bortezomib-mediated killing of melanoma cells, the apoptotic pathway involving NOXA was further investigated, leading to identification of an important target (i.e., the labile Bcl-2 homologue Mcl-1 but not other survival proteins). To reduce Mcl-1 levels, melanoma cells were pretreated with several different agents, including Mcl-1 small interfering RNA (siRNA), UV light, or the purine nucleoside analogue fludarabine. By simultaneously triggering production of NOXA (using bortezomib) as well as reducing Mcl-1 levels (using siRNA, UV light, or fludarabine), significantly enhanced killing of melanoma cells was achieved. These results show binding interactions between distinct Bcl-2 family members, such as NOXA and Mcl-1, in melanoma cells, paving the way for novel and rational therapeutic combination strategies, which target guardians of the proapoptotic Bak- and Bax-mediated pathways, against this highly aggressive and often fatal malignancy.

Defining the transcriptome of accelerated and replicatively senescent keratinocytes reveals links to differentiation, interferon signaling, and Notch related pathways.

Epidermal keratinocytes (KCs) undergo highly orchestrated morphological and molecular changes during transition from proliferative compartment into growth arrested early and late differentiation layers, prior to dying in outermost cornified layers of normal skin. Creation of stratum corneum is vital to barrier function protecting against infection. Transcriptional events in KCs regulating complex processes of differentiation and host defense required to maintain constant epidermal thickness and resistance to infection in either young or aged skin are largely unknown. Furthermore, as terminal differentiation is characterized by irreversible loss of replicative potential culminating in dead layers at the skin surface, this process may be viewed as a form of senescence. However, a complete transcriptional profile of senescent (SN) human KCs has not been previously defined to permit delineation of molecular boundaries involving differentiation and senescence. To fill this void, we utilized global transcriptional analysis of KCs maintained in vitro as either cultures of proliferating (PR) cells, early and late confluent (LC) (accelerated senescence) cultures, or KCs undergoing replicative senescence. Global gene expression profiling revealed early confluent (EC) KCs were somewhat similar to PR KCs, while prominent differences were evident when compared to LC KCs; which were also distinct from replicatively SN KCs. While confluent KCs have in common several genes regulating differentiation with replicatively SN KCs, the latter cells expressed elevated levels of genes involved in interferon signaling and inflammatory pathways. These results provide new insights into cell autonomous transcriptional-based programs operative within KCs contributing to replicative senescence, with partial sharing of genes involved in differentiation. In addition, regulation of KC senescence may involve participation of interferon signaling pathways derived from the important role of KCs in protecting skin from infection. Integrating all of the transcriptional data revealed a key role for Notch receptor mediated signaling in the confluency induced differentiation phenotype using this model system.

Notch and NOXA-related pathways in melanoma cells.

Notch receptor-mediated intracellular events represent an ancient cell signaling system, and alterations in Notch expression are associated with various malignancies in which Notch may function as an oncogene or less commonly as a tumor suppressor. Notch signaling regulates cell fate decisions in the epidermis, including influencing stem cell dynamics and growth/differentiation control of cells in skin. Because of increasing evidence that the Notch signaling network is deregulated in human malignancies, Notch receptors have become attractive targets for selective killing of malignant cells. Compared with proliferating normal human melanocytes, melanoma cell lines are characterized by markedly enhanced levels of activated Notch-1 receptor. By using a small molecule gamma-secretase inhibitor (GSI) consisting of a tripeptide aldehyde, N-benzyloxycarbonyl-Leu-Leu-Nle-CHO, which can block processing and activation of all four different Notch receptors, we identified a specific apoptotic vulnerability in melanoma cells. GSI triggers apoptosis in melanoma cells, but only G2/M growth arrest in melanocytes without subsequent cell death. Moreover, GSI treatment induced a pro-apoptotic BH3-only protein, NOXA, in melanoma cells but not in normal melanocytes. The use of GSI to induce NOXA induction overcomes the apoptotic resistance of melanoma cells, which commonly express numerous cell survival proteins such as Mcl-1, Bcl-2, and survivin. Taken together, these results highlight the concept of synthetic lethality in which exposure to GSI, in combination with melanoma cells overexpressing activated Notch receptors, has lethal consequences, producing selective killing of melanoma cells, while sparing normal melanocytes. By identifying signaling pathways that contribute to the transformation of melanoma cells (e.g. Notch signaling), and anti-cancer agents that achieve tumor selectivity (e.g., GSI-induced NOXA), this experimental approach provides a useful framework for future therapeutic strategies in cutaneous oncology.

Pathways involved in proliferating, senescent and immortalized keratinocyte cell death mediated by two different TRAIL preparations.

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.