Powerful Skin Cancer Protection by a CPD-Photolyase Transgene

AbstractBackground: The high and steadily increasing incidence of ultraviolet-B (UV-B)-induced skin cancer is a problem recognized worldwide. UV introduces different types of damage into the DNA, notably cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs). If unrepaired, these photolesions can give rise to cell death, mutation induction, and onset of carcinogenic events, but the relative contribution of CPDs and 6-4PPs to these biological consequences of UV exposure is hardly known. Because placental mammals have undergone an evolutionary loss of photolyases, repair enzymes that directly split CPDs and 6-4PPs into the respective monomers in a light-dependent and lesion-specific manner, they can only repair UV-induced DNA damage by the elaborate nucleotide excision repair pathway.Results: To assess the relative contribution of CPDs and 6-4PPs to the detrimental effects of UV light, we generated transgenic mice that ubiquitously express CPD-photolyase, 6-4PP-photolyase, or both, thereby allowing rapid light-dependent repair of CPDs and/or 6-4PPs in the skin. We show that the vast majority of (semi)acute responses in the UV-exposed skin (i.e., sunburn, apoptosis, hyperplasia, and mutation induction) can be ascribed to CPDs. Moreover, CPD-photolyase mice, in contrast to 6-4PP-photolyase mice, exhibit superior resistance to sunlight-induced tumorigenesis.Conclusions: Our data unequivocally identify CPDs as the principal cause of nonmelanoma skin cancer and provide genetic evidence that CPD-photolyase enzymes can be employed as effective tools to combat skin cancer

Long-term in vitro 2D-culture of SDHB and SDHD-related human paragangliomas and pheochromocytomas

The neuroendocrine tumours paraganglioma and pheochromocytoma (PPGLs) are commonly associated with succinate dehydrogenase (SDH) gene variants, but no human SDHrelated PPGL-derived cell line has been developed to date. The aim of this study was to systematically explore practical issues related to the classical 2D-culture of SDH-related human paragangliomas and pheochromocytomas, with the ultimate goal of identifying a viable tumour-derived cell line. PPGL tumour tissue/cells (chromaffin cells) were cultured in a variety of media formulations and supplements. Tumour explants and dissociated primary tumour cells were cultured and stained with a range of antibodies to identify markers suitable for use in human PPGL culture. We cultured 62 PPGLs, including tumours with confirmed SDHB, SDHC and SDHD variants, as well as several metastatic tumours. Testing a wide range of basic cell culture media and supplements, we noted a marked decline in chromaffin cell numbers over a 4-8 week period but the persistence of small numbers of synaptophysin/ tyrosine hydroxylase-positive chromaffin cells for up to 99 weeks. In cell culture, immunohistochemical staining for chromogranin A and neuron-specific enolase was generally negative in chromaffin cells, while staining for synaptophysin and tyrosine hydroxylase was generally positive. GFAP showed the most consistent staining of type II sustentacular cells. Of the media tested, low serum or serum-free media best sustained relative chromaffin cell numbers, while lactate enhanced the survival of synaptophysin-positive cells. Synaptophysin- positive PPGL tumour cells persist in culture for long periods but show little evidence of proliferation. Synaptophysin was the most consistent cell marker for chromaffin cells and GFAP the best marker for sustentacular cells in human PPGL cultures

A novel mouse model for Sezary Syndrome using xenotransplantation of Sezary cells into immunodeficient RAG2-/-gamma c-/- mice

Isoprenylcysteine (IPC) molecules modulate G-protein-coupled receptor signalling. The archetype of this class is N-acetyl-S-farnesyl-l-cysteine (AFC). Topical application of AFC locally inhibits skin inflammation and elicitation of contact hypersensitivity in vivo. However, the mechanism of these anti-inflammatory effects is not well understood. Dermal microvascular endothelial cells (ECs) are involved in inflammation, in part, by secreting cytokines that recruit inflammatory cells. We have previously shown that the sympathetic nerve cotransmitter adenosine-5'-triphosphate (ATP) and adenosine-5'-O-(3-thio) triphosphate (ATP?S), an ATP analogue that is resistant to hydrolysis, increase secretion of the chemokines CXCL8 (interleukin-8), CCL2 (monocyte chemotactic protein-1) and CXCL1 (growth-regulated oncogene a) by dermal microvascular ECs. Production of these chemokines can also be induced by the exposure to the proinflammatory cytokine TNFa. We have now demonstrated that AFC dose-dependently inhibits ATP-, ATP?S- and TNFa-induced production of CXCL1, CXCL8 and CCL2 by a human dermal microvascular EC line (HMEC-1) in vitro under conditions that do not affect cell viability. Inhibition of ATP?S- or TNFa-stimulated release of these chemokines was associated with reduced mRNA levels. N-acetyl-S-geranyl-l-cysteine, an IPC analogue that is inactive in inhibiting G-protein-coupled signalling, had greatly reduced ability to suppress stimulated chemokine production. AFC may exert its anti-inflammatory effects through the inhibition of chemokine production by stimulated EC

Comparison of five cell culture media.

Tested on Tu44 (thoracic PPGL, SDHD p.(D92Y)) and Tu46 (carotid body PGL, SDHD p.(L95P)), stained with 3 marker antibodies and scored double blinded in duplo. Percentages of positive cells, with standard error of the mean (SEM), are presented in 10log scale. P values calculated with one-way ANOVA (p S1 Table for details) when sufficient tissue was available. We assessed cultures for the presence of synaptophysin as a marker for chromaffin cells and GFAP as a marker for sustentacular cells. In addition, we estimated the proportion of proliferating cells based on Ki-67 expression. In general, serum-free medium supported the highest proportion of synaptophysin-positive cells, but no long-term serum-free cultures including these cells were noted, probably because serum-free culture medium leads to the slow depletion of cells. Chromaffin cells visibly fail to prosper in this medium, often typified by poor filopodia development and poor adhesion. Media including some bovine serum, at either 1% or 5%, seemed to achieve a better balance between proliferation of fibroblasts, which may have a supportive function in these cultures, and survival of chromaffin tumour cells. Richer media such as Izal and PC12 led to a predominance of fibroblasts that appeared detrimental to the survival of chromaffin cells, possibly due to the rapid consumption of available metabolites in the media.</p

Tumour explant culture to determine the ongoing cellularity of explants.

Tumour explants (1x1 mm tumour fragments) were cultured for up to 39 days to assess cellularity and remaining chromaffin tumour cells. At day 1, H&E staining of tumour 9 reveals the classic cell nest morphology in a highly stromal background (A, Tu9_CBT_SDHD_H&E_10x obj.). By around day 11 tumour fragments began to show clear signs of declining cellularity and an increase in eosin-avid (pink) acellular proteinaceous material (S2 Fig). By day 39, few cells of chromaffin appearance remained in the tumour fragments (B, Tu9_CBT_SDHD_H&E_10x obj.). Chromaffin cellularity was determined using immunohistochemistry (C, Tu44_ thoracic PGL_SDHD_Syn_10x obj.) to assess the proportion of synaptophysin-positive cells (‘chromaffin’ tumour cells) remaining at 1 week (C, Tu44, CBT_SDHD_Syn_10x obj.). At around 4 weeks, (D, Tu44_ thoracic PGL_SDHD_Syn_10x obj.) declining expression of synaptophysin (brown staining) was accompanied by visible shrinkage of cellular tumour areas. Residual chromaffin cell areas can be seen but the lack of nucleic acid (DNA) staining by haematoxylin suggests a loss of cellular integrity. Areas lightly staining for synaptophysin probably consist of cellular debris of chromaffin cells. Quantification of synaptophysin immunohistochemistry in three tumours (E, TU40-CBT, Tu42-PPGL, Tu43-CBT) showed a consistent decline in the expression of this marker protein in explants over a 4-week culture period.</p

Summary of immunocytochemical staining of PPGL tumour cultures.

Both chromogranin A (Tu8_Chromogranin A_10x obj.) and neuron-specific enolase (Tu-28_Neuron-specific enolase (NSE)_10x obj.) are generally negative, even in very early cultures. Tyrosine hydroxylase staining (Tu36_Tyrosine hydroxylase (TH)_40x obj.) is often positive, even in long-term cultures, but as many HNPGL cultures are negative for TH at the outset this marker is not generally applicable. In our hands the most broadly reliable marker in PPGL tumour cultures proved to be synaptophysin (Tu18_Syn_40x obj.), as this marker can be found in both early and late cultures. To assess the survival of neuronal cells in PPGL tumour cell cultures we also stained for neurofilament protein (Tu-7_Neurofilament protein_20x obj.), which is expressed in nerve fibres and the sparse individual neurons seen in PPGL tumours. We found that even at 29 months of culture (Tu7), a few cells of neuronal morphology are still present in culture. CD56 (NCAM) is a cell surface marker for chromaffin cells and is found in most PPGLs, showing the distinct staining pattern of a cell surface protein. Of the few tumour cultures examined (Tu-42_CD56 (ab 123C3) _10x obj.), only sparse cells were positive for CD56. The classic markers for sustentacular cells in PPGLs are S100 and GFAP. S100 tends to show more widespread staining of these cells and is therefore favoured in IHC applications, while GFAP is expressed more sporadically in the same cells. Both marker proteins are expressed in tumour cultures but S100 expression (Tu-40_S100_20x obj.) appears to be short-lived, while GFAP expression (Tu44_thoracic PGL_GFAP_20x obj.) persists somewhat longer, both generally disappearing over the course of 4–8 weeks, along with cells showing sustentacular morphology. CD31 is a marker for endothelial cells and staining of PPGL FFPE tumour sections underscores the extensive vascularity of these tumours. Although few cultures were stained with CD31, those that were appeared uniformly negative (Tu-8_CD31_20x obj.), suggesting that endothelial cells do not persist in culture. Ki-67, a protein expressed in proliferating cells, was occasionally positive in cells of fibroblast morphology (Tu-52_Ki-67_20x obj.). Cytokeratin, a marker for epithelial cells, was occasionally positive (Tu-8_Cytokeratin_10x obj.) in a few cells. Cytoskeleton proteins such as smooth muscle actin (SMA), vimentin and fibronectin are present in most cells and as such are aspecific markers. However, they can usefully illustrate the general morphology of cell cultures. We found SMA (Tu-47_Smooth muscle actin (SMA)_40x obj._cytospin) and vimentin (Tu-28_Vimentin_10x obj.;Tu-8_Vimentin_10x obj.) to be the most useful, with fibronectin only sporadically positive in a few cells (Tu-17 _Fibronectin_20x obj.).</p

Antibody stainings of formalin-fixed paraffin-embedded (FFPE) PPGL tumors.

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Surgically excised PPGLs vary considerably in their gross appearance.

Pliant, highly vascular tumours (A) digest readily, whereas firm, dark tumours (B) generally require more rigorous treatment to release tumour cells. Digested tissue features single cells and large and small groups of tumour cells (C, Tu57_10x obj. phase contrast). Tumour-derived cells show an irregular light halo around a darker central area, in contrast to the ‘doughnut’ appearance of erythrocytes (D, Tu57_40x obj. phase contrast). Long-term cultures occasionally develop areas that show roughly circular lattice structures (E, Tu11_4 months_5% FBS_10x obj.) that resemble sustentacular cell networks found in tumours but more likely represent connective tissue cells specific to PPGLs, perhaps suggesting that these cells have self-organizing capabilities specific to this tissue. Paraganglioma cell cultures may also develop extensive networks of processes (F, Tu21_4 months_5% FBS_5x obj.), usually but not always after an extended period in culture. These networks can become macroscopically visible in cell culture flasks and usually interconnect discrete cell masses. Cells of tumour culture 23, a paraganglioma without a variant in any commonly-mutated gene, exhibited short, eccentrically branching cell processes (G, Tu23_20 days_5% FBS_20x obj.), occasionally accumulating around a single, flattened cell. Tumour cells originating from a PPGL bone metastasis showed a semi-differentiated morphology in culture (H, Tu26_6 days_5% FBS_10x obj.), a morphology occasionally seen in other non-metastatic tumours.</p

Summary of the gene variants found in this study.

Abbreviations: SDHB, C & D, succinate dehydrogenase subunits B, C & D, respectively; HNPGL, head & neck paraganglioma; PPGL, pheochromocytoma-sympathetic paraganglioma; meta, metastatic.</p

Immunocytochemical scoring of control cell lines and a range of PGL tumour cultures based on morphological criteria.

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