C-Terminal Farnesylation of UCH-L1 Plays a Role in Transport of Epstein-Barr Virus Primary Oncoprotein LMP1 to Exosomes.

Increasing evidence shows that exosomes are key regulators in cancer cell-to-cell communication. Several reports on Epstein-Barr virus (EBV)-related malignancies demonstrate that latent membrane protein 1 (LMP1) secreted by exosomes derived from EBV- or LMP1-positive cells can promote cancer progression and metastasis. However, the mechanism by which LMP1 is loaded into exosomes is still poorly understood. Here, we examined whether the process of LMP1 loading into exosomes is linked to the multifunctional molecule of the ubiquitin system-ubiquitin C-terminal hydrolase-L1 (UCH-L1). For the first time, we demonstrate that LMP1 is physically associated with UCH-L1 and that directing of LMP1 to exosomes is mediated by C-terminal farnesylation of UCH-L1. Additionally, we found that the FTI-277 farnesyltransferase inhibitor reduces motility- and anchorage-independent growth of EBV-positive cells in functional assays. On the basis of our results, we conclude that C-terminal farnesylation of UCH-L1 is one of the key mechanisms by which LMP1 is sorted to exosomes. We hypothesize that inhibition of farnesylation with specific small-molecule inhibitors blocks exosome-mediated transfer of prometastatic molecules such as LMP1 during cancer cell-to-cell communications and thereby impedes the process of cancer invasion. IMPORTANCE Exosomes are small vesicles that cells secrete into the extracellular space, and there is increasing evidence that they have pivotal roles in cell-to-cell communication in malignancy. It is reported also that EBV-associated malignant cells, including those derived from nasopharyngeal carcinoma (NPC) and B-cell lymphoma, secrete exosomes. These EBV-related exosomes may contain viral products such as latent membrane protein 1 (LMP1) and may contribute to cancer progression. The aim of this study was to investigate the mechanism by which those viral products are loaded in exosomes. In this study, we show for the first time that ubiquitin C-terminal hydrolase-L1 (UCH-L1) and its C-terminal farnesylation, a posttranslational lipid modification, contribute to this mechanism. Our results also suggest that inhibition of UCH-L1 farnesylation is a potential therapeutic target against cancer metastasis and invasion.

Photodynamic therapy for superficial basal cell carcinoma and Bowen's disease.

Photodynamic therapy (PDT) is an effective treatment for superficial basal cell carcinoma (BCCs) and Bowen's disease. Several studies have reported complete response rates of 80-95% and an excellent cosmetic outcome. Bowen's disease and superficial basal cell carcinomas characteristically affect older patients who may also have associated difficulties with mobility. Using a portable PDT light source we were able to deliver PDT in a community setting with the aim of providing a more convenient service for patients. This randomised study confirmed that community delivered PDT is a viable treatment option and can be administered safely in the community by a trained dermatology nurse. The results from patient questionnaires suggest that community delivered PDT is more convenient to the patient, and also cost effective.

An open study to determine the efficacy of blue light in the treatment of mild to moderate acne.

BACKGROUND: The effective management of acne remains a challenge; achieving an optimal response whilst minimizing adverse events is often difficult. The rise in antibiotic resistance threatens to reduce the future usefulness of the current mainstay of therapy. The need for alternative therapies remains important. Phototherapy has previously been shown to be effective in acne, with renewed interest as both endogenous and exogenous photodynamic therapies are demonstrated for this condition. OBJECTIVES: To determine the effect of narrowband blue light in the reduction of inflammatory and non-inflammatory lesions in patients with mild to moderate acne and to evaluate patient tolerance of the therapy. METHODS: We performed an open study utilizing a blue LED light source in 30 subjects with mild to moderate facial acne. Two weeks after screening, lesions were counted and recorded by lesion type. Over 4 weeks, patients received eight 10- or 20-minute light treatments, peak wavelength 409-419 nm at 40 mW/cm2. Assessments were taken at weeks 5, 8 and 12 and lesion counts were recorded. Repeated measures-ANOVA and Dunnett's tests, respectively, allowed assessment of the different scores over time and permitted comparison of mean counts. RESULTS: An overall effect on inflammatory counts was observed at week 5, and a statistically significant decrease in inflamed counts was detected at the week 8 assessments, which continued to week 12. There was little effect on non-inflamed lesions. The treatment was well tolerated with adverse events experienced generally rated as being mild and usually self-limiting. CONCLUSIONS: Eight 10- or 20-minute treatments over 4 weeks with a narrowband blue light was found to be effective in reducing the number of inflamed lesions in subjects with mild to moderate acne. The treatment had little effect on the number of comedones. The onset of the effect was observable at the first assessment, at week 5, and maximal between weeks 8 and 12. Blue light phototherapy using a narrowband LED light source appears to be a safe and effective additional therapy for mild to moderate acne.

Comparison of the cellular molecular stress responses after treatments used in bladder cancer.

OBJECTIVE: To investigate the molecular stress responses related to the quality of recovery of normal tissue after various treatments for bladder cancer, i.e. hyperthermia, ionizing radiation, mitomycin-C and 5-aminolaevulinic acid photodynamic therapy (ALA-PDT). MATERIALS AND METHODS: The study focused particularly on intracellular fibroblast levels of heat-shock protein-47 (HSP47) and HSP72, which are associated with collagen metabolism and the development of tolerance to repeated treatment, respectively. Iso-effective treatment doses (50% clonogenic cell survival) of each method were delivered to a 3T6 murine fibroblast model. Intracellular extracts were analysed at 3, 6, 9, 12 and 24 h after treatment, using Western blot analysis to compare the levels of HSP47 and HSP72. Time-matched treatment and control groups were quantified by comparison with actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression using appropriate software. RESULTS: There were various changes in levels of HSP expression with treatment method; HSP47 levels were significantly higher after hyperthermia and radiation but not with mitomycin-C or ALA-PDT. HSP72 levels were significantly higher with all methods except ALA-PDT. CONCLUSIONS: Hyperthermia and ionizing radiation are associated with early increases in levels of HSP47 (a marker of collagen metabolism), in contrast to ALA-PDT and mitomycin-C. These findings are compatible with clinical findings where fibrosis/scarring is common with the first two but not the last two methods. In addition, all methods except ALA-PDT are associated with an increase in HSP 72 (a protein associated with cellular tolerance) and this may help to explain, at a cellular level, why resistance to repeated ALA-PDT treatments does not seem to occur.

Photodynamic therapy for superficial bladder cancer under local anaesthetic.

OBJECTIVES: To evaluate the use of local anaesthesia (LA) in 5-aminolaevulinic acid (ALA) photodynamic therapy (PDT) for superficial transitional cell carcinoma (TCC) of the bladder, and to provide further toxicity and tolerability data on this new method within the context of a phase 1 trial. PATIENTS AND METHODS: ALA PDT was administered to 19 patients with recurrent superficial TCC (stage Ta/carcinoma in situ, grades 1-3) using escalating doses of ALA (3-6%) and 633 nm laser light (25-50 J/cm2) under various LA (lignocaine) protocols. Pain was assessed using a linear analogue scale from 0 to 10. The endpoints of tolerability and toxicity were assessed for the different LA, light and ALA doses, with lignocaine levels. RESULTS: ALA PDT is painful and requires some form of anaesthesia. The discomfort was immediate, associated with bladder spasm, and was a function of the ALA concentration rather than the total light dose given. Simple passive diffusion (PD) of 2% lignocaine instilled for 40 min before PDT gave adequate anaesthesia with 3% ALA (n=8; median pain score 1, range 0-2). With 6% ALA the pain was dramatically increased using PD (n=6; median pain score 8, range 5-10) and therefore the more potent LA technique of electromotive drug administration (EMDA) of 2% lignocaine was used, with excellent results (n=3; median pain score 1, range 0-2). All patients had transient bladder irritability that typically lasted 9-12 days, with no subjective/objective change in long-term bladder function. No other toxicity was reported. Serum lignocaine levels were minimal. CONCLUSION: Bladder ALA PDT is both safe and feasible under LA. At a dose of 3% ALA, the procedure was well-tolerated using PD of lignocaine. At higher doses (6% ALA) more effective anaesthesia is required and this can be obtained satisfactorily with EMDA of lignocaine. With refinement, ALA PDT may be feasible as an outpatient treatment for superficial bladder TCC.

Normal TCRbeta transcription and recombination in the absence of the Jbeta2-Cbeta2 intronic cis element.

The developmental regulation of antigen receptor gene transcription and recombination are mediated by cis regulatory elements. At the T cell receptor beta chain locus (TCRbeta), two DNase I hypersensitive sites within the Jbeta2-Cbeta2 intron contained binding sites for NF-kappaB and additional nuclear factors and were postulated to be involved in controlling TCRbeta transcription and V(D)J recombination. To test this possibility, we deleted these elements from the mouse genome by homologous recombination and assayed the effect on transcription of both the germline and rearranged TCRbeta locus, and on TCRbeta rearrangement in T and B lymphocytes. We found that TCRbeta transcription and V(D)J recombination and T cell development were normal in these mutant mice. Therefore, the Jbeta2-Cbeta2 intronic elements are dispensable for TCRbeta assembly and function.

Photodynamic therapy for large or multiple patches of Bowen disease and basal cell carcinoma.

BACKGROUND: Photodynamic therapy (PDT) using topical delta-aminolevulinic acid (delta-ALA) is an effective treatment for Bowen disease and certain basal cell carcinomas (BCCs), but its place in clinical practice remains to be established. Patients with large and/or multiple lesions of Bowen disease or BCC can represent a considerable therapeutic challenge. We suggest that delta-ALA PDT may be of particular benefit in such patients. OBSERVATION: In an open study, 35 (88%) of 40 large patches of Bowen disease, all with a maximum diameter greater than 20 mm, cleared following 1 to 3 treatments of delta-ALA PDT, although 4 patches recurred within 12 months. delta-Aminolevulinic acid PDT was also used to treat 40 large BCCs, with an identical 88% initial clearance (after 1-3 treatments), with 4 recurrences within 34 months (range, 12-60 months). In 10 further patients with multiple (> or =3) patches of Bowen disease, 44 (98%) of 45 patches cleared following delta-ALA PDT, although 4 lesions recurred over 12 months. In 3 patients with multiple BCCs, PDT cleared 52 (90%) of 58 lesions, with 2 recurrences during 41 months (range, 12-52 months). Treatments were well tolerated, with only 5 patients with solitary large lesions requiring local anesthesia. CONCLUSIONS: delta-Aminolevulinic acid PDT is an effective tissue-sparing modality achieving good cosmesis. We propose that delta-ALA PDT be considered as a first-line therapy for large and/or multiple areas of Bowen disease and superficial BCCs.

Photodynamic therapy for superficial bladder cancer.

In photodynamic therapy, a photosensitizing drug is activated by visible light and in the presence of oxygen, results in local cell death. This evolving modality is now being used to treat and palliate a very wide variety of human solid tumors and carcinoma-in-situ lesions. With regard to bladder cancer, advances in drug development and modern light delivery techniques mean that photodynamic therapy shows promise in the treatment of superficial bladder cancer resistant to conventional treatments.

Routine double treatments of superficial basal cell carcinomas using aminolaevulinic acid-based photodynamic therapy.

BACKGROUND: Superficial basal cell carcinomas of the skin (sBCC) often respond poorly to single-treatment aminolaevulinic acid-based photodynamic therapy (ALA-PDT), with a number of reports indicating a relapse rate of 50% or more. OBJECTIVES: To determine whether a second treatment at seven days can improve the response. METHODS: Twenty-six lesions were treated twice with ALA-PDT, with an interval of 7 days between the two treatment sessions. RESULTS: We observed a complete response rate of 100% 1 month after treatment. Only one lesion relapsed (16 months post-PDT), a relapse rate of 4% (median follow up 27 months; range 15-45 months). Cosmetic results were excellent. CONCLUSIONS: We consider routine double treatments with ALA-PDT to be an effective approach to the management of sBCC, particularly those located in anatomically difficult, or cosmetically sensitive, sites.

Deletion of germline promoter PD beta 1 from the TCR beta locus causes hypermethylation that impairs D beta 1 recombination by multiple mechanisms.

The role of the germline transcriptional promoter, PD beta 1, in V(D)J recombination at the T cell receptor beta locus was investigated. Deletion of PD beta 1 caused reduced germline transcription and DNA hypermethylation in the Dbeta1-J beta 1 region and decreased D beta 1 rearrangement. Analyses of methylation levels surrounding recombination signal sequences (RSS) before, during, and after recombination revealed that under physiological conditions cleavage of hypomethylated alleles was preferred over hypermethylated alleles. Methylation of a specific CpG site within the heptamer of the 3' D beta 1 RSS was incompatible with cleavage by the V(D)J recombinase. These findings suggest that methylation can regulate V(D)J recombination both at a general level by influencing regional chromatin accessibility and specifically by blocking RSS recognition or cleavage by the V(D)J recombinase.

Comparison of red and green light in the treatment of Bowen's disease by photodynamic therapy.

BACKGROUND: A variety of protocols exist for the treatment of Bowen's disease by photodynamic therapy (PDT) using topical 5-aminolaevulinic acid (5-ALA). OBJECTIVE: To determine the optimal wavelength (red or green light) for this treatment. METHODS: A randomized comparison study of ALA-PDT using red (630 +/- 15 nm) or green (540 +/- 15 nm) light in the treatment of Bowen's disease. RESULTS: The initial clearance rate for lesions treated by red light was 94% (30 of 32) in comparison with 72% (21 of 29) for those lesions receiving green light (P = 0.002). Over the following 12 months, there were two recurrences in the red light group and seven in the green light group reducing the clearance rates to 88% and 48%, respectively. The frequency and severity of pain experienced were similar between the two treatment groups. No hyperthermia, nor significant difference in lesional temperatures, was observed between the wavelengths studied. CONCLUSION: Green light is less effective than red light, at a theoretically equivalent dose, in the treatment of Bowen's disease by topical ALA-PDT.

Light penetration in bladder tissue: implications for the intravesical photodynamic therapy of bladder tumours.

OBJECTIVES: To assess (i) the optical properties and depth of penetration of varying wavelengths of light in ex-vivo human bladder tissue, using specimens of normal bladder wall, transitional cell carcinoma (TCC) and bladder tissue after exposure to ionizing radiation; and (ii) to estimate the depth of bladder wall containing cancer that could potentially be treated with intravesical photodynamic therapy (PDT), assuming satisfactory tissue levels of photosensitizer. Materials and methods The study included 11 cystectomy specimens containing invasive TCC (five from patients who had previously received external-beam bladder radiotherapy, but with recurrent TCC) and three 'normal' bladders removed from patients treated by exenteration surgery for extravesical pelvic cancer. Full-thickness bladder wall and tumour samples were taken from these specimens and using an 'intravesical' and a previously validated interstitial model, the optical penetration depths (i.e. the tissue depth at which the light fluence is 37% of incident) were calculated at wavelengths of 633, 673 and 693 nm. RESULTS: There were no significant differences in light penetration between normal and tumour-affected bladder tissue at each wavelength. There were significant differences in light penetration among wavelengths; light at 693 nm penetrated approximately 40% further than light at 633 nm (P < 0.002). The light currently used in bladder PDT (633 nm) has a mean (SEM) optical penetration depth of 4.0 (0.1) mm within TCC. In addition, at this wavelength, there was 29% greater light penetration in previously irradiated than in unirradiated bladder wall (P = 0.001). This did not occur in the tumour-affected bladder. CONCLUSIONS: Bladder tissue is relatively more translucent than other human tissues and there is therefore great potential for PDT in the treatment of bladder cancer. As there is no difference in light penetration between TCC and normal bladder tissue, a tumour-specific response with diffuse illumination of the bladder will depend on drug localization within the tumour. The currently used wavelength of 633 nm can be expected to exert a PDT effect within bladder tumour up to a depth of 20 mm. Increasing the wavelength will allow deeper pathology to be treated.

An interstitial light assembly for photodynamic therapy in prostatic carcinoma.

OBJECTIVE: To develop an interstitial laser light delivery system using multiple optical fibres for photodynamic therapy (PDT) in the treatment of prostate cancer. PATIENTS AND METHODS: A laser beam was divided equally with a 1 x 4 fibre splitter to deliver PDT simultaneously through four 2-cm long, flexible cylindrical optical diffusers. Biplanar transrectal ultrasonography (TRUS) and a template were used to position the optical fibres percutaneously. In vivo measurements of light penetration depth (1/micro[eff] ) in prostate tissue were made in seven patients, using a sheathed isoprobe to measure light fluence rates at varying radial distances from the diffuser. The prostate was fixed with stabilization needles to minimize displacement during needle placement. RESULTS: The mean (sd, range) micro(eff) in the prostates of the seven patients was 0.35 (0.07, 0.22-0.44) mm-1, which produced closely parallel slopes of light attenuation. However, there was up to a 10-fold variation in absolute light levels at the same diffuser-detector separation distances amongst the seven patients, probably caused by blood pooling around the diffuser light source. A similar problem around the isoprobe detector was overcome by sheathing the probe in clear plastic tubing. By stabilizing the prostate, the optical fibre positioning was precise to within 2 mm. CONCLUSION: Although this light delivery and TRUS assembly were developed for clinical PDT in the prostate, the same instrumentation can be used reliably for in vivo light-penetration studies. Haemorrhage was unpredictable and highlighted one of the main problems which needs to be overcome.

Control of V(D)J recombinational accessibility of the D beta 1 gene segment at the TCR beta locus by a germline promoter.

The germline promoter region upstream of the D beta 1 gene segment in the murine TCR beta locus was deleted to assess its role in controlling V(D)J recombination. Associated with diminished D beta 1 region germline transcription, rearrangement of the D beta 1 but not the D beta 2 gene segment was reduced 10- to 20-fold. A corresponding reduction in RAG-mediated cleavage at the D beta 1 and J beta 1 signal sequences was apparent only when purified CD4- CD8- thymocytes were analyzed because, as we demonstrate, cleavage at these gene segments also occurred in CD4+ CD8+ thymocytes. These findings suggest that germline promoters regulate localized accessibility of gene segments for recombination and that in CD4+ CD8+ thymocytes TCR beta allelic exclusion does not result from inaccessibility of D beta gene segments.

T lymphocyte development in the absence of CD3 epsilon or CD3 gamma delta epsilon zeta.

CD3 gamma, delta, epsilon, and zeta proteins together with the pre-TCR alpha-chain (pT alpha) and a rearranged TCR beta-chain assemble to form the pre-TCR that controls the double negative (DN) to double positive (DP) stages of thymopoiesis. The CD3 proteins are expressed before pT alpha and TCR beta-chains in prothymocytes and are expressed intracellularly in precursor NK cells, suggesting that the CD3 complex may function independent of pT alpha and TCR beta. In this report, both the role of CD3 epsilon exclusively, and the role of CD3 proteins collectively, in thymocyte and NK cell development were examined. In a mouse strain termed E delta P, a neomycin cassette inserted within the CD3 epsilon promoter abolishes CD3 epsilon and delta expression and also abolishes CD3 gamma expression in all but a small minority (< or =1%) of prothymocytes. These prothymocytes became deficient in CD3 epsilon alone upon reconstitution of CD3 delta expression and were severely, but not completely, arrested at the DN stage, as small numbers of double positive thymocytes were detected. In de facto CD3 gamma delta epsilon zeta(null) mice generated by crossing the epsilon delta P mice with CD3 zeta-/- mice, thymopoiesis were arrested at the CD44-CD25+ DN stage as observed in RAG-/- mice, DJ and VDJ recombination at the TCR beta locus was functional, and normal numbers of NK cells were detected. Together, the findings demonstrate that during thymocyte development, the CD3 complex collectively is not essential until the critical CD44-CD25+ DN stage in which pre-TCR begins to function, whereas CD3 epsilon is critical for the assembly of pre-TCR. Moreover, CD3 proteins are dispensable for NK cell development.

A nuclear matrix attachment region upstream of the T cell receptor beta gene enhancer binds Cux/CDP and SATB1 and modulates enhancer-dependent reporter gene expression but not endogenous gene expression.

We have previously identified a DNase I-hypersensitive site in the T cell receptor beta locus, designated HS1, that is located 400 base pairs upstream of the transcriptional enhancer Ebeta and is induced during CD4(-)CD8(-) to CD4(+)CD8(+) thymocyte differentiation. Using electrophoretic mobility shift assays, we show that HS1 induction correlates with increased binding of two nuclear factors, Cux/CDP and SATB1, to a 170-base pair DNA sequence within HS1. Furthermore, we demonstrate that HS1 is a nuclear matrix attachment region, referred to as MARbeta. These findings demonstrate that an analogous organization of cis-regulatory elements in which a nuclear matrix attachment region is in close proximity to an enhancer is conserved in the immunoglobulin and T cell receptor loci. In addition, we show that MARbeta represses Ebeta-dependent reporter gene expression in transient transfection assays. However, the targeted deletion of MARbeta from the endogenous locus does not change T cell receptor beta gene transcription in developing T cells. These contrasting results suggest a potential pitfall of functional studies of nuclear matrix attachment regions outside of their natural chromosomal context.