Consequences of depleted SERCA2-gated calcium stores in the skin

Sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) pumps belong to the family of Ca2+-ATPases responsible for the maintenance of calcium in the endoplasmic reticulum. In epidermal keratinocytes, SERCA2-controlled calcium stores are involved in cell cycle exit and onset of terminal differentiation. Hence, their dysfunction was thought to provoke impaired keratinocyte cohesion and hampered terminal differentiation. Here, we assessed cultured keratinocytes and skin biopsies from a canine family with an inherited skin blistering disorder. Cells from lesional and phenotypically normal areas of one of these dogs revealed affected calcium homeostasis due to depleted SERCA2-gated stores. In phenotypically normal patient cells, this defect compromised upregulation of p21(WAF1) and delayed the exit from the cell cycle. Despite this abnormality it failed to impede the terminal differentiation process in the long term but instead coincided with enhanced apoptosis and appearance of chronic wounds, suggestive of secondary mutations. Collectively, these findings provide the first survey on phenotypic consequences of depleted SERCA-gated stores for epidermal homeostasis that explain how depleted SERCA2 calcium stores provoke focal lesions rather than generalized dermatoses, a phenotype highly reminiscent of the human genodermatosis Darier disease.

Expression and regulation of interleukins in human head and neck squamous cell carcinoma

Patients with head and neck squamous cell carcinoma (HNSCC) demonstrate abnormal cell-mediated immunity which is most pronounced at the primary tumor site. Therefore, we tested whether this aberrant immunity could be due to tumor-derived cytokines. We investigated the presence of cytokine mRNA and protein in 8 HNSCC-derived cell lines; RT-PCR results indicated mRNA's for IL-1$\alpha$ and TGF-$\alpha$ (8/8), TGF-$\beta$ (7/8), IL-1$\beta$ (7/8), IL-4 and IL-6 (4/8). IL-2, IFN-$\gamma,$ and TNF-$\alpha$ mRNA was not detected. Supernatants from 6 of these cell lines were analyzed by ELISA and IL-1$\alpha,$ IL-1$\beta,$ and IL-6 were markedly increased compared to HPV-16 immortalized human oral keratinocytes. IL-1$\alpha$ was found in the highest concentration $>$IL-6 $>$ IL-1$\beta.$^ To approach the mechanisms of cytokine regulation, 4 cell lines were compared for HPV DNA presence, p53 status, and cytokine expression. An association between HPV DNA and cytokine expression was not found. However, cell lines secreting the most IL-6 had mutant p53 and/or HPV 16 E6/E7 expression. Further regulatory investigations revealed that exogenous IL-1$\alpha$ and/or IL-1$\beta$ minimally stimulated the proliferation of 2/3 cell lines, as well as strongly induced IL-6 production in 3/3; this effect was completely abrogated by IL-1Ra. IL-1Ra also inhibited the secretion of IL-1$\alpha$ and IL-1$\beta$ in 2/3 cell lines. These data suggest an IL-1 autocrine loop in certain HNSCC cell lines. Because IL-2 induces IL-1 and is used in therapy of HNSCC, the expression of IL-2 receptor was also investigated; IL-2 $\alpha$ and $\beta$ subunits were detected in 3/3 cell lines and $\gamma$ subunits was detected in one. Exogenous IL-2 inhibited the proliferation, but stimulated the secretion of IL-1$\alpha$ in 2/3, and IL-1$\beta$ and IL-6 in 1/3 cell lines.^ To determine if our cell line findings were applicable to patients, immunohistochemistry was performed on biopsies from 12 invasive tumors. Unexpectedly, universal intracellular production of IL-1$\alpha,$ IL-1$\beta,$ and IL-6 protein was detected. Therefore, the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients. ^

Regulation of angiogenesis by interferon-beta

The purpose of these studies was to investigate the role of interferon-beta (IFN-$\beta$) in angiogenesis. IFN-$\alpha/\beta$ have been implicated in inhibiting a number of steps in the angiogenic pathway. We examined the balance of angiogenesis-regulating molecules in several systems including human infantile hemangiomas, UV-B irradiated mice, and dorsal incisional wound healing in mice. In each system, epidermal hyperplasia and cutaneous angiogenesis were directly related to the expression of positive angiogenic factors (bFGF and VEGF) and inversely related to the expression of endogenous IFN-$\beta.$ The re-expression of IFN-$\beta$ correlated with tumor regression and/or resolution of wound healing. In contrast to control mice, UV-B-induced cutaneous angiogenesis and hyperplasia persisted in IFN-$\alpha/\beta$ receptor knock-out mice. In normal mice, endogenous IFN-$\beta$ was expressed by all differentiated epithelial cells exposed to environmental stimuli. The expression of endogenous IFN-$\beta$ was necessary but insufficient for complete differentiation of epidermal keratinocytes.^ The tumor organ microenvironment can regulate angiogenesis. Human bladder carcinoma cells growing in the bladder wall of nude mice express high levels of bFGF, VEGF, and MMP-9, have higher vascular densities, and produce metastases to lymph nodes and lungs, whereas the same cells growing subcutaneously express less bFGF, VEGF, and MMP-9, have lower vascular densities, and do not metastasize. IFN-$\alpha/\beta$ was found to inhibit bFGF and MMP-9 expression both in vitro and in vivo in human bladder carcinoma cells. Systemic therapy with human IFN-$\alpha$ of human bladder cancer cells growing orthotopically in nude mice, resulted in decreased vascularity, tumorigenicity, and metastasis as compared to saline treated mice. Human bladder cancer cells resistant to the antiproliferative effects of IFN were transfected with the human IFN-$\beta$ gene. Hu-IFN-$\beta$ transfected cells expressed significantly less bFGF protein and gelatinase activity than parental or control-transfected cells and did not grow at ectopic or orthotopic sites. Collectively the data provide direct evidence that IFN-$\alpha/\beta$ can inhibit angiogenesis via down-regulation of angiogenesis-stimulating cytokines. ^

Examination of synthetic retinoid -induced apoptosis in cutaneous squamous cell carcinomas: Mechanisms and implications for chemoprevention and chemotherapy with retinoids

Non-melanoma skin cancers, including basal cell carcinoma and squamous cell carcinoma (SCC), are the most common neoplasms in the United States with a lifetime risk nearly equal to all other types of cancer combined. Retinoids are naturally occurring and synthetic analogues of vitamin A that bind to nuclear retinoid receptors and modulate gene expression as a means of regulating cell proliferation and differentiation. Retinoids have been employed for many years in the treatment of various cutaneous lesions and for cancer chemoprevention and therapy. The primary drawback limiting the use of retinoids is their toxicity, which is also associated with receptor-gene interactions. In this study, the effects of the synthetic retinoids N-(4-hydroxyphenyl)retinamide (4HPR) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) were examined in cutaneous keratinocytes. Four human cutaneous SCC cell lines were examined along with normal human epidermal keratinocyte (NHEK) cells from two donors. Sensitivity to 4HPR or CD437 alone or in combination with other agents was determined via growth inhibition, cell cycle distributions, or apoptosis induction. Both synthetic retinoids were able to promote apoptosis in SCC cells more effectively than the natural retinoid all-trans retinoic acid. Apoptosis could not be inhibited by nuclear retinoic acid receptor antagonists. In NHEK cells, 4HPR induced apoptosis while CD437 promoted G1 arrest. 4HPR acted as a prooxidant by generating reactive oxygen species (ROS) in SCC and NHEK cells. 4HPR-induced apoptosis in SCC cells could be inhibited or potentiated by manipulating cellular defenses against oxidative stress, indicating an essential role for ROS in 4HPR-induced apoptosis. CD437 promoted apoptosis in SCC cells in S and G2/M phases of the cell cycle within two hours of treatment, and this rapid induction could not be blocked with cycloheximide. This study shows: (1) 4HPR- and CD437-induced apoptosis do not directly involve a traditional retinoid pathway; (2) 4HPR can act as a prooxidant as a means of promoting apoptosis; (3) CD437 induces apoptosis in SCC cells independent of protein synthesis and is potentially less toxic to NHEK cells; and (4) 4HPR and CD437 operate under different mechanisms with respect to apoptosis induction and this may potentially enhance their therapeutic index in vivo. ^

Mouse Deformed epidermal autoregulatory factor 1 recruits a LIM domain factor, LMO-4, and CLIM coregulators

Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, we used the conserved LIM-interaction domain of Clim coregulators to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and isolated a factor that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, we used LMO-4 as a bait to screen for such DNA-binding proteins in epidermis and isolated the mouse homologue of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix–loop–helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain.

Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes.

Cellular senescence is defined by the limited proliferative capacity of normal cultured cells. Immortal cells overcome this regulation and proliferate indefinitively. One step in the immortalization process may be reactivation of telomerase activity, a ribonucleoprotein complex, which, by de novo synthesized telomeric TTAGGG repeats, can prevent shortening of the telomeres. Here we show that immortal human skin keratinocytes, irrespective of whether they were immortalized by simian virus 40, human papillomavirus 16, or spontaneously, as well as cell lines established from human skin squamous cell carcinomas exhibit telomerase activity. Unexpectedly, four of nine samples of intact human skin also were telomerase positive. By dissecting the skin we could show that the dermis and cultured dermal fibroblasts were telomerase negative. The epidermis and cultured skin keratinocytes, however, reproducibly exhibited enzyme activity. By separating different cell layers of the epidermis this telomerase activity could be assigned to the proliferative basal cells. Thus, in addition to hematopoietic cells, the epidermis, another example of a permanently regenerating human tissue, provides a further exception of the hypothesis that all normal human somatic tissues are telomerase deficient. Instead, these data suggest that in addition to contributing to the permanent proliferation capacity of immortal and tumor-derived keratinocytes, telomerase activity may also play a similar role in the lifetime regenerative capacity of normal epidermis in vivo.

In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.

Human papillomavirus (HPV) types 16, 18, 31, and 51 are the etiologic agents of many anogenital cancers including those of the cervix. These "high risk" HPVs specifically target genital squamous epithelia, and their lytic life cycle is closely linked to epithelial differentiation. We have developed a genetic assay for HPV functions during pathogenesis using recircularized cloned HPV 31 genomes that were transfected together with a drug resistance marker into monolayer cultures of normal human foreskin keratinocytes, the natural host cell. After drug selection, cell lines were isolated that stably maintained HPV 31 DNA as episomes and underwent terminal differentiation when grown in organotypic raft cultures. In differentiated rafts, the expression of late viral genes, amplification of viral DNA, and production of viral particles were detected in suprabasal cells. This demonstrated the ability to synthesize HPV 31 virions from transfected DNA templates and allowed an examination of HPV functions during the vegetative viral life cycle. We then used this system to investigate whether an episomal genome was required for the induction of late viral gene expression. When an HPV 31 genome (31E1*) containing a missense mutation in the E1 open reading frame was transfected into normal human keratinocytes, the mutant viral sequences were found to integrate into the host cell chromosomal DNA with both early and late regions intact. While high levels of early viral gene transcription were observed, no late gene expression was detected in rafts of cell lines containing the mutant viral genome despite evidence of terminal differentiation. Therefore, the induction of late viral gene expression required that the viral genomes be maintained as extrachromosomal elements, and terminal differentiation alone was not sufficient. These studies provide the basis for a detailed examination of HPV functions during viral pathogenesis.

Ligand-directed retroviral targeting of human breast cancer cells.

We explored the feasibility of designing retroviral vectors that can target human breast cancer cells with characteristic receptors via ligand-receptor interaction. The ecotropic Moloney murine leukemia virus envelope was modified by insertion of sequences encoding human heregulin. Ecotropic virus, which normally does not infect human cells, when pseudotyped with the modified envelope protein now crosses species to infect human breast cancer cell lines that overexpress HER-2 (human epidermal growth factor receptor; also called ERBB2) and HER-4 (also called ERBB4), while human breast cancer cell lines expressing low levels of these receptors remain resistant to infection. Since about 20% of human breast cancers overexpress HER-2 and some of breast cancer cell lines overexpress both HER-2 and HER-4, cell-specific targeting of retroviral vectors may provide a different approach for in vivo gene therapy of this type of breast cancer.

Characterization of tissue transglutaminase 2 in macrophage function

Apoptosis is a highly regulated process that removes damaged or unwanted cells in vivo and defective clearance of apoptotic cells by macrophages has significant immunological implications. Tissue transglutaminase 2 (TG2) is a Ca2+-dependent protein cross linking enzyme known to play an important role in cell proliferation, differentiation, carcinogenesis, programmed death, and aging. TG2 as a guanosine triphosphate (GTP)-binding or GTP- hydrolyzing protein for mediating signal transduction and as a cell cycle regulator emphasized the importance of this enzyme in aging process. The ubiquitous presence of TG2 compared to the other organ-specific TGases has attracted special attention as a cellular aging device. TG2 activity and expression are known to increase in aging humans suggesting possible involvement in several age-related processes such as decrease in vascular compliance and increased stiffening of conduit arteries, cataract formation, Alzheimer's disease and senescent epidermal keratinocytes. Our work aims to characterize the role of TG2 and its partners (e.g. syndecan-4 and ß3 integrin) in macrophage function. THP-1 cell derived macrophage-like cells and primary human macrophages were analyzed for the expression and function of TG2. Macrophage-apoptotic cell interaction studies in the presence of TG2 inhibitors resulted in significant inhibition of interaction. Macrophage cell surface TG2 and, in particular, its cell surface cross linking activity was found to be crucial in apoptotic cell clearance. Syndecan-4 association with TG2 implies possible cooperation of these proteins and knockdown studies of syndecan-4 reveal its importance in apoptotic cell clearance. Our current findings suggest that TG2 has a crucial but yet to be fully defined role in apoptotic cell clearance.

A co-cultured skin model based on cell support membranes

Tissue engineering of skin based on collagen:PCL biocomposites using a designed co-culture system is reported. The collagen:PCL biocomposites having collagen:PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen:PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen:PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen:PCL biocomposite membranes for preparation of a bi-layered skin model with differentiated epidermal sheet was proven in principle. The approach to skin modeling reported here may find application in tissue engineering and screening of new pharmaceuticals. © 2005 Elsevier Inc. All rights reserved.

Endothelin 3 induces skin pigmentation in a keratin-driven inducible mouse model

Endothelin 3 (Edn3) is a ligand important to developing neural crest cells (NCC). Some NCC eventually migrate into the skin and give rise to the pigment-forming melanocytes found in hair follicles. Edn3's effects on NCC have been largely explored through spontaneous mutants and cell culture experiments. These studies have shown the Endothelin receptor B/Edn3 signaling pathway to be important in the proliferation/survival and differentiation of developing melanocytes. To supplement these investigations I have created doxycycline-responsive transgenic mice which conditionally over-express Edn3. These mice will help us clarify Edn3's role during the development of early embryonic melanoblasts, differentiating melanocyte precursors in the skin, and fully differentiated melanocytes in the hair follicle. The transgene mediated expression of Edn3 was predominantly confined to the roof plate of the neural tube and surface ectoderm in embryos and postnatally in the epidermal keratinocytes of the skin. Relative to littermate controls, transgenics develop increased pigmentation on most areas of the skin. My doxycycline-based temporal studies have shown that both embryonic and postnatal events are important for establishing and maintaining pigmented skin. The study of my Edn3 transgenic mice may offer some insight into the genetics behind benign dermal pigmentation and offer clues about the time periods important in establishing these conditions. This apparently abnormal development is echoed in a benign condition of human skin. Cases of dermal melanocytosis, such as common freckles, Mongolian spotting, and nevus of Ito demonstrate histological and etiological characteristics similar to those of the transgenic mice generated in this study.

A new future for growth factor complexes as wound therapies?

Background: Topical administration of growth factors (GFs) has displayed some potential in wound healing, but variable efficacy, high doses and costs have hampered their implementation. Moreover, this approach ignores the fact that wound repair is driven by interactions between multiple GFs and extracellular matrix (ECM) proteins. The Problem: Deep dermal partial thickness burn (DDPTB) injuries are the most common burn presentation to pediatric hospitals and also represent the most difficult burn injury to manage clinically. DDPTB often repair with a hypertrophic scar. Wounds that close rapidly exhibit reduced scarring. Thus treatments that shorten the time taken to close DDTPB’s may coincidently reduce scarring. Basic/Clinical Science Advances: We have observed that multi-protein complexes comprised of IGF and IGF-binding proteins bound to the ECM protein vitronectin (VN) significantly enhance cellular functions relevant to wound repair in human skin keratinocytes. These responses require activation of both the IGF-1R and the VN-binding αv integrins. We have recently evaluated the wound healing potential of these GF:VN complexes in a porcine model of DDTPB injury. Clinical Care Relevance: This pilot study demonstrates that GF:VN complexes hold promise as a wound healing therapy. Enhanced healing responses were observed after treatment with nanogram doses of the GF:VN complexes in vitro and in vivo. Critically healing was achieved using substantially less GF than studies in which GFs alone have been used. Conclusion: These data suggest that coupling GFs to ECM proteins, such as VN, may ultimately prove to be an improved technique for the delivery of novel GF-based wound therapies.

Safety and efficacy of the combination of trastuzumab with docetaxel for HER2-positive women with advanced breast cancer : a review of the existing clinical trials and results of the expanded access programme in the UK

Trastuzumab is a humanised monoclonal antibody against the extracellular domain of HER2 (human epidermal growth factor receptor-2) that is overexpressed in about 25% of human breast cancers. It has shown clinical benefit in HER2-positive breast cancer cases when used alone or in combination with chemotherapy. Trastuzumab increases the response rate to chemotherapy and prolongs survival when used in combination with taxanes. In this article, we review the clinical trials where trastuzumab has been administered together with docetaxel, and we present the results of the trastuzumab expanded access programme (EAP) in the UK. Combination of trastuzumab with docetaxel results in similar response rates and time-to-progression with the trastuzumab/paclitaxel combinations. The toxicity of the combination and the risk of heart failure are low. The clinical data for the docetaxel/trastuzumab combination indicate a favourable profile from both the efficacy and the safety point of view and confirm the feasibility and safety of trastuzumab administration both as monotherapy and in combination with docetaxel. © 2004 Blackwell Publishing Ltd.

Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations

Purpose The LUX-Lung 3 study investigated the efficacy of chemotherapy compared with afatinib, a selective, orally bioavailable ErbB family blocker that irreversibly blocks signaling from epidermal growth factor receptor (EGFR/ErbB1), human epidermal growth factor receptor 2 (HER2/ErbB2), and ErbB4 and has wide-spectrum preclinical activity against EGFR mutations. A phase II study of afatinib in EGFR mutation-positive lung adenocarcinoma demonstrated high response rates and progression-free survival (PFS). Patients and Methods In this phase III study, eligible patients with stage IIIB/IV lung adenocarcinoma were screened for EGFR mutations. Mutation-positive patients were stratified by mutation type (exon 19 deletion, L858R, or other) and race (Asian or non-Asian) before two-to-one random assignment to 40 mg afatinib per day or up to six cycles of cisplatin plus pemetrexed chemotherapy at standard doses every 21 days. The primary end point was PFS by independent review. Secondary end points included tumor response, overall survival, adverse events, and patient-reported outcomes (PROs). Results A total of 1,269 patients were screened, and 345 were randomly assigned to treatment. Median PFS was 11.1 months for afatinib and 6.9 months for chemotherapy (hazard ratio [HR], 0.58; 95% CI, 0.43 to 0.78; P = .001). Median PFS among those with exon 19 deletions and L858R EGFR mutations (n = 308) was 13.6 months for afatinib and 6.9 months for chemotherapy (HR, 0.47; 95% CI, 0.34 to 0.65; P = .001). The most common treatmentrelated adverse events were diarrhea, rash/acne, and stomatitis for afatinib and nausea, fatigue, and decreased appetite for chemotherapy. PROs favored afatinib, with better control of cough, dyspnea, and pain. Conclusion Afatinib is associated with prolongation of PFS when compared with standard doublet chemotherapy in patients with advanced lung adenocarcinoma and EGFR mutations.

Alternating current electrohydrodynamics induced nanoshearing and fluid micromixing for specific capture of cancer cells

We report a new tuneable alternating current (ac) electrohydrodynamics (ac-EHD) force referred to as “nanoshearing” which involves fluid flow generated within a few nanometers of an electrode surface. This force can be externally tuned via manipulating the applied ac-EHD field strength. The ability to manipulate ac-EHD induced forces and concomitant fluid micromixing can enhance fluid transport within the capture domain of the channel (e.g., transport of analytes and hence increase target–sensor interactions). This also provides a new capability to preferentially select strongly bound analytes over onspecifically bound cells and molecules. To demonstrate the utility and versatility of nanoshearing phenomenon to specifically capture cancer cells, we present proof-of-concept data in lysed blood using two microfluidic devices containing a long array of asymmetric planar electrode pairs. Under the optimal experimental conditions, we achieved high capture efficiency (e.g., approximately 90%; %RSD=2, n=3) with a 10-fold reduction in nonspecific dsorption of non-target cells for the detection of whole cells expressing Human Epidermal Growth Factor Receptor 2 (HER2). We believe that our ac-EHD devices and the use of tuneable nanoshearing phenomenon may find relevance in a wide variety of biological and medical applications.