Enzyme defects in xeroderma pigmentosum

Fibroblast strains were obtained from 12 patients with xeroderma pigmentosum of various clinical types. Repair replication of UV-damaged DNA in the fibroblasts was studied by 3H-thymidine labeling and radioautography. DNA repair replication was found decreased in all xeroderma pigmentosum fibroblasts compared with control cells obtained from normal donors. Repair activities in patients cells ranged from nearly 0% in three infant cases and two cases of De Sanctis-Cacchione syndrome to approximately 100% in an adult moderate case. There was, however, no correlation between the level of repair replication and the severity of clinical symptoms. Since three cases which showed a lack of repair DNA replication were infants, it is assumed that these cases may develop De Sanctis-Cacchione syndrome in the future. A genetic analysis of xeroderma pigmentosum cells, was performed with cell fusion methods using irradiated HVJ virus in order to determine the type of the complementation group. XP-1, XP-3, XP-4, XP-6 and XP-9 may be classified into group D; XP-2, XP-7, XP-8, XP-11 and XP-12 into group A; and XP-5 into the group E.     

De Sanctis-Caccione syndrome: xeroderma pigmentosum with oligophrenia, short stature and neurologic disorders]

A 6 year-old boy with de Sanctis-Cacchione syndrome is reported. This syndrome is characterized by the triad xeroderma pigmentosum (XP), mental deficiency and neurological disturbances. The patient's cells were assigned to genetic complementation group A by use of the cell fusion technique. DNA repair capacity as measured by unscheduled DNA synthesis (UDS) was drastically reduced to 7.5%, compared with 100% of the controls. The rate of sister chromatid exchange (SCE), an indicator of the hypermutability in XP, was clearly elevated after ultraviolet radiation of skin fibroblasts of the patient.     

DNA mismatch repair protein Msh6 is required for optimal levels of ultraviolet-B-induced apoptosis in primary mouse fibroblasts

Recent data support a role for DNA mismatch repair in the cellular response to some forms of exogenous DNA damage beyond that of DNA repair; cells with defective DNA mismatch repair have partial or complete failure to undergo apoptosis and/or G2M arrest following specific types of damage. We propose that the DNA mismatch repair Msh2/Msh6 heterodimer, responsible for the detection of DNA damage, promotes apoptosis in normal cells, thus protecting mammals from ultraviolet-induced malignant transformation. Using primary mouse embryonic fibroblasts derived from Msh6+/+ and Msh6-/- mice, we compare the response of DNA-mismatch repair-proficient and -deficient cells to ultraviolet B radiation. In the wild-type mouse embryonic fibroblasts, ultraviolet-B-induced increases in Msh6 protein levels were not dependent on p53. Msh6-/- mouse embryonic fibroblasts were significantly less sensitive to the cytotoxic effects of ultraviolet B radiation. Further comparison of the Msh6+/+ and Msh6-/- mouse embryonic fibroblasts revealed that Msh6-/- mouse embryonic fibroblasts undergo significantly less apoptosis following ultraviolet B irradiation, thus indicating that ultraviolet-B-induced apoptosis is partially Msh6 dependent. These data support a role for Msh6 in protective cellular responses of primary cells to ultraviolet-B-induced mutagenesis and, hence, the prevention of skin cancer.     

SASP‐induced macrophage dysfunction may contribute to accelerated senescent fibroblast accumulation in the dermis

Recently, increasing attention has been paid to senescence‐associated secretory phenotype (SASP), a phenomenon that senescent cells secrete molecules such as inflammatory cytokines and matrix metalloproteinases (MMPs), due to its noxious effects on the surrounding tissue. Senescent cells in the blood and liver are known to be properly depleted by macrophages. In the dermis, accumulation of senescent cells has been reported and is thought to be involved with skin ageing. In this study, to elucidate the clearance mechanism of senescent cells in the dermis, we focused on macrophage functions. Our co‐culture experiments of senescent fibroblasts and macrophages revealed a two‐step clearance mechanism: first, TNF‐α secreted from macrophages induces apoptosis in senescent fibroblasts, and then, dead cells are phagocytosed by macrophages. Furthermore, it was suggested that SASP factors suppress both of the two steps of the senescent cell clearance by macrophages. From these findings, normally senescent cells in the dermis are thought to be removed by macrophages, but when senescent cells are excessively accumulated owing to oxidative stress, ultraviolet (UV) ray or other reasons, SASP was suggested to suppress the macrophage‐dependent clearance functions and thereby cause further accumulation of senescent cells.     

De Sanctis-Cacchione syndrome

We present a male patient with photosensitivity since the earliest months of his life, and pigmented macules in exposed areas, some showing clinical atypia, which increased in number over time. Molecular biology studies detected an alteration in DNA repair ability, so xeroderma pigmentosum was diagnosed. Shortly after birth, low weight, microcephaly and psychomotor retardation had been observed, but the cause was not established. The patient progressively showed neurological disorders that included perceptive deafness, hyporeflexia and areflexia, as well as choreoathetotic movements. Therefore, we felt that the patient's symptoms fit De Sanctis-Cacchione syndrome.     

The De Barsy syndrome

BACKGROUND: In 1968, De Barsy reported on a girl exhibiting an aged aspect, 'dwarfism, oligophrenia, and degeneration of the elastic tissue in cornea and skin'. The disorder was recognized as a subgroup of cutis laxa syndrome and termed De Barsy-Moens-Dierckx syndrome. The pathogenesis of the disorder is unknown. METHODS: To improve the comprehension of the pathogenetic mechanisms involved in the De Barsy syndrome, we performed an ultrastructural, morphometric, immunocytochemical study on a skin biopsy of a boy with the De Barsy phenotype, who has been clinically followed for 12 years from birth. Moreover, the lysyl oxidase activity was measured on skin fibroblasts cultured in vitro. RESULTS: Light and electron microscopy, morphometry, and immunocytochemical observations showed a significant reduction of the elastic fibers in the papillary and in the reticular dermis of patient compared to an age-matched control (p < 0.05). By contrast, the collagen structure, content, and the distribution were normal, as well as lysyl oxidase activity in the medium of in vitro fibroblasts (12,323 DPM/10(6) cells). The immunoreaction for antibodies recognizing fibrillin-1, neutrophilic elastase, and tumor necrosis factor-alpha was stronger, whereas that for antibodies against transforming growth factor-beta was less pronounced in the dermis of the De Barsy boy compared to control. CONCLUSIONS: Clinical, phenotypic, and structural data were consistent with the diagnosis of De Barsy syndrome. This is the first case described in Italy. Clinical and structural data confirm that the elastic component is mostly affected in this disorder. Moreover, ultrastructural and immunochemical findings suggest that both elastic fiber degradative and very likely synthetic processes are involved.     

Replicative senescence of human dermal fibroblasts affects structural and functional aspects of the Golgi apparatus

It is well recognized that the world population is ageing rapidly. Therefore, it is important to understand ageing processes at the cellular and molecular levels to predict the onset of age‐related diseases and prevent them. Recent research has focused on the identification of ageing biomarkers, including those associated with the properties of the Golgi apparatus. In this context, Golgi‐mediated glycosylation of proteins has been well characterized. Additionally, other studies show that the secretion of many compounds, including pro‐inflammatory cytokines and extracellular matrix–degrading enzymes, is modified during ageing, resulting in physical and functional skin degradation. Since the Golgi apparatus is a central organelle of the secretory pathway, we investigated its structural organization in senescent primary human dermal fibroblasts using confocal and electron microscopy. In addition, we monitored the expression of Golgi‐related genes in the same cells. Our data showed a marked alteration in the Golgi morphology during replicative senescence. In contrast to its small and compact structure in non‐senescent cells, the Golgi apparatus exhibited a large and expanded morphology in senescent fibroblasts. Our data also demonstrated that the expression of many genes related to Golgi structural integrity and function was significantly modified in senescent cells, suggesting a relationship between Golgi apparatus function and ageing.     

A new disorder in UV-induced skin cancer with defective DNA repair distinct from xeroderma pigmentosum or Cockayne syndrome

We report the characterization of a Japanese woman who exhibited many freckles and skin cancers in sun-exposed areas, but displayed no photosensitivity. Fibroblasts (KPSX7) derived from this patient showed similar UV sensitivity to that of normal human fibroblasts. The KPSX7 cells showed normal levels of unscheduled DNA synthesis, recovery of RNA synthesis, recovery of replicative DNA synthesis, protein-binding ability to UV-damaged DNA, and post-translational modification of xeroderma pigmentosum (XP) C. These results indicate that the patient had neither XP nor Cockayne syndrome. Although these results suggest that the KPSX7 cells were proficient in nucleotide excision repair activity, host-cell reactivation (HCR) activity of KPSX7 cells was reduced. Furthermore, introduction of UV damage endonuclease into the cells restored repair activity in the HCR assay to almost normal levels. These results indicate that KPSX7 cells are defective for some types of repair activity in UV-damaged DNA. In summary, the patient had a previously unknown disorder related to UV-induced carcinogenesis, with defective DNA repair.     

Radiobiological studies in the naevoid basal cell carcinoma syndrome

The naevoid basal cell carcinoma syndrome is described in a 43-year-old male. The response of dividing fibroblasts from this patient to the lethal effects of ionizing radiation was indistinguishable from that of a normal control: mean inactivation dose (D) = 1.74 Gy and 1.68 Gy, respectively. However, these cells were defective in the repair of potentially lethal damage (D = 2.36 Gy and 3.26 Gy, respectively). The response to radiation of Go T lymphocytes derived from the patient was similar to that of the control. The frequency of mutant circulating T lymphocytes resistant to 6-thioguanine was 17 x 10(-6) which was elevated when compared with age-matched controls. These results suggest an abnormality in the response of the fibroblasts to ionizing radiation damage.     

De Sanctis-Cacchione Syndrome in a female infant - Case report

The De Sanctis-Cacchione Syndrome is the rarest and most severe kind of xeroderma pigmentosum, characterized by microcephaly, hypogonadism, neurological disorders, mental and growth retardation, with very few cases published. The clinical findings compatible with De Sanctis-Cacchione Syndrome and the therapeutic approach used to treat a one year and nine months old child, with previous diagnosis of xeroderma pigmentosum, are reported.     

UVB应激性衰老成纤维细胞分泌细胞外基质对HaCaT细胞ERK信号的影响

目的 探讨UVB诱导的衰老（UVB-SIPS）成纤维细胞分泌的细胞外基质（ECM）对HaCaT细胞增殖的影响以及细胞外信号调节激酶（ERK）信号途径的作用。方法 采用辐射诱导人皮肤成纤维细胞衰老,分别制备由未衰老和衰老成纤维细胞分泌的ECM包被的培养皿（分别定为PRE-ECM组、SCIP-ECM组）。以空白培养皿（NON-ECM组）为参照,在HaCaT细胞接种于培养皿后,采用MTT法和流式细胞法检测其细胞增殖、细胞周期等指标的变化;免疫印迹法分析ERK活化水平。干预性研究ERK信号通路对上述功能的影响。结果 三组中,SCIP-ECM组可诱导最强烈的ERK1/2的活化。采用U0126抑制ERK信号活化可完全抑制衰老成纤维细胞来源的ECM的促细胞增殖效应。阻断HaCaT细胞的ERK活化,NON-ECM组、PRE-ECM组和SCIP-ECM组S + G2/M期细胞的比例明显下降,分别由处理前37.40%、41.34%和43.31%减少至29.41%、36.48%到39.96%。结论 UVB-SCIP成纤维细胞分泌的ECM通过刺激ERK磷酸化促进HaCaT细胞增殖。     

X-linked dyskeratosis congenita with pancytopenia

Two maternal male cousins in a Jewish Iraqi kindred were affected with dyskeratosis congenita and had a megaloblastic bone marrow. One cousin had pancytopenia and the other had thrombocytopenia. The kindred displays a deficiency of glucose-6-phosphate dehydrogenase (G6PD) and a beta-thalassemia trait. The following genetic "markers" of the X chromosome were studied: G6PD, the X-linked blood groups Xg, and color vision. Linkage analysis indicated that dyskeratosis, G6PD, and Xg are far apart on the X chromosome. Chromosomal studies showed a 46XY karyotype in both cases; however, nonspecific numerical aberrations and structural abnormalities were found in the first and in the second case, polyploidy was seen in four of 60 cells. The proband's cultured fibroblasts did not show increased susceptibility to malignant transformation by simian virus 40, an oncogenic virus.     

Phenotypic and functional changes in dermal primary fibroblasts isolated from intrinsically aged human skin

Dermal fibroblasts play a key role in maintaining skin homoeostasis by synthesizing and degrading extracellular matrix components. During ageing, they are subjected to changes, such as the loss of type I collagen expression and an increased synthesis of metalloproteinase I, leading to fragmentation of collagen fibrils with consequent reduction of the mechanical tension and defects of skin wound healing. Most information about fibroblast ageing was obtained from experiments performed on replicative‐senescent dermal fibroblasts in vitro. However, the senescence status of fibroblasts isolated from intrinsically aged skins and its consequences on functionality need to be deeper investigated. Herein, we studied age‐related phenotypic and functional alteration of fibroblasts from ‘young’ (<35 years) and ‘old’ (>50 years) donors. Our results brought evidence of the senescent status of ‘old’ fibroblasts by senescence associated β‐galactosidase (SA‐βgal) positive staining and p16 expression. A PCR array focusing on senescence highlighted a subset of downregulated genes including cell cycle progression and ECM genes in ‘old’ fibroblasts as well as a subset of upregulated genes involved in senescence features. In ‘old’ fibroblasts, we measured a downregulation of proliferative and contractile capacities of migratory potential under PDGF stimulation and activation into myofibroblasts under TGFβ. Old fibroblasts were also more sensitive to oxidative stress than ‘young’ ones. Of interest, downregulation of p16 expression partially reversed the senescent phenotype of ‘old’ fibroblasts but failed to restore their functional properties. In conclusion, our data brought evidence of phenotypic and functional differences between fibroblasts from young and intrinsically aged skin that may contribute to the alterations observed with ageing.     

Enhanced expression of p16 in seborrhoeic keratosis; a lesion of accumulated senescent epidermal cells in G1 arrest

BACKGROUND: Seborrhoeic keratosis (SK) is a common skin disease associated with skin ageing and photoageing, but only limited studies have been performed on SK and the senescence of keratinocytes. OBJECTIVES: We sought to clarify the genetic basis of SK and the senescence of keratinocytes. METHODS: Expression of p16, cyclins A, D and E, p21, p53, retinoblastoma (Rb) gene product and telomerase-associated protein 1 (TP1) in SK was examined by immunohistochemistry. DNA fragmentation in SK was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling method. We cultured keratinocytes from SK lesions and non-lesional epidermis and examined expression of p16, observed morphology of the cultured cells by light and electron microscopy and measured survival time. RESULTS: p16, a cyclin-dependent kinase inhibitor, was expressed in all cells from SK lesions, whereas normal keratinocytes expressed p16 only in the granular cells. Other factors such as cyclins A, D and E, p21, p53, Rb gene product, and TP1, were not expressed in SK cells. These results suggest that p16 expression is a marker of SK and that p16 has a role in the pathogenesis of SK. DNA fragmentation was not detected in four of five SK tissue samples; one of the SK tissue samples showed DNA fragmentation only in the superficial upper layer of an SK lesion, suggesting that apoptosis was inhibited in SK cells. In contrast, normal epidermis showed DNA fragmentation in the granular and squamous layers. Immunohistochemical examination of cultured SK cells also revealed the presence of p16. A greater number of SK cells survived after 3 weeks of culture in comparison with normal keratinocytes. Features of senescence, such as a balloon-like appearance after lengthy culture and increased amounts of tonofilaments in cytoplasm, were observed in SK cells in culture. CONCLUSIONS: These results suggest that SK is a benign neoplasm where keratinocytes in a senescent condition and G1 arrest are accumulated.     

葡萄糖-6-磷酸脱氢酶的表达抑制对A431细胞增殖和细胞周期的影响

目的研究葡萄糖?6?磷酸脱氢酶（G6PD）表达下调对皮肤鳞状细胞癌（鳞癌）细胞增殖和细胞周期的影响。方法正常培养人永生化上皮细胞HaCaT、皮肤鳞癌SCL?1和A431细胞,采用Western印迹法检测细胞中G6PD蛋白的表达。当A431细胞生长至85%~90%融合时,将siRNA对照（siRNA对照组）和G6PD siRNA（G6PD siRNA组）分别转染A431细胞,未转染的A431细胞则为未转染组。Western印迹法检测3组不同处理的A431细胞中G6PD蛋白及细胞周期蛋白D1、CDK4的表达,CCK?8法检测3组A431细胞的增殖情况,流式细胞仪分析3组A431细胞周期的变化。结果正常培养的2株皮肤鳞癌SCL?1和A431细胞中G6PD蛋白的表达水平（分别为0.308±0.023和0.643±0.046）均显著高于HaCaT细胞（0.100±0.019）,且A431细胞显著高于SCL?1细胞（均P<0.05）。A431细胞G6PD siRNA组G6PD、细胞周期蛋白D1和CDK4蛋白的表达（0.134±0.027、0.154±0.017、0.166±0.017）显著低于未转染组（0.425±0.029、0.344±0.024、0.330±0.020）和siRNA对照组（0.444±0.033、0.350±0.027、0.348±0.018）,差异均有统计学意义（P<0.05）。G6PD siRNA组在24~96 h各时间点的细胞增殖活性均明显低于siRNA对照组和未转染组（P<0.001）,而siRNA对照组与未转染组间细胞增殖差异无统计学意义（均P>0.05）。G6PD siRNA组G0/G1期A431细胞比例显著高于siRNA对照组及未转染组（P<0.001）,而G6PD siRNA组S期A431细胞比例又显著低于siRNA对照组及未转染组（P<0.001）。结论G6PD可能在调控皮肤鳞癌细胞增殖和细胞周期中发挥重要作用。     

Senescent dermal fibroblasts enhance stem cell migration through CCL2/CCR2 axis

During aging, increases in the number of senescent cells are seen in various tissues. On the other hand, stem cells play crucial roles in tissue repair and homeostasis. Therefore, it is likely that stem cells give rise to new cells that replace senescent cells. However, how stem cells contribute to homeostasis in the dermis has not been elucidated. Here, we investigated the effects of factors secreted from senescent fibroblasts on stem cells. We found that senescent human dermal fibroblast (HDF) conditioned medium (CM) significantly enhanced stem cell migration compared with young HDF CM. The senescent HDF CM strongly secreted chemokine ligand 2 (CCL2). Furthermore, CCL2 was found to enhance stem cell migration, and the inhibition of CCR2, a receptor for CCL2, reduced stem cell migration. These results suggest that senescent fibroblasts recruit stem cells by secreting various factors and that the CCL2/CCR2 axis is one of the mechanisms underlying this phenomenon.     

Senescent fibroblasts in melasma pathophysiology

It has been proposed that melasma is a photoageing skin disorder. The photoaged fibroblasts have been suggested as an important source of melanogenic factors which are involved in the regulation of pigmentation. To investigate whether melasma includes senescent cells, lesional and perilesional normal skin from 38 melasma patients was assessed using a cell senescence marker, p16^INK4A. The results showed that lesional dermal skin had more p16^INK4A‐positive senescent cells than perilesional skin. The impact of senescent fibroblasts was further investigated in a pilot study using radiofrequency (RF) intervention for melasma. It showed that the RF therapy decreased the number of senescent cells with increased expression of procollagen‐1, which were associated with reduced epidermal pigmentation. This leads us to the speculation that senescent fibroblasts may contribute to drive melasma and might be considered as a potential therapeutic target.