Cooperation of Ha-ras and Bcl-2 during multistep skin carcinogenesis

Nonmelanoma skin cancer (NMSC) is the most frequently diagnosed cancer in the United States. Deregulation of bcl-2 and ras family members is commonly observed in NMSC. It has been previously demonstrated that simultaneous bcl-2 and Ha-ras gene expression in keratinocytes results in disordered differentiation and resistance to cell death induced by ultraviolet (UV) radiation. It was, therefore, interest to assess the extent of cooperation between bcl-2 and Ha-ras during multistep skin carcinogenesis. The keratin 1 promoter was used to generate HK1.ras and HK1.bcl-2 transgenic mice, which were subsequently crossed to generate HK1.ras/bcl-2 double transgenic mice. The apoptotic index (AI) following UV-irradiation was significantly lower in HK1.bcl-2 and HKI.ras/bcl-2 epidermis compared to control littermates. Interestingly, the AI of HK1.ras/bcl-2 mice was significantly lower than even HK1.bcl-2 mice following UV-irradiation. To investigate the interaction of these oncogenes in skin tumorigenesis, a two-stage chemical carcinogenesis protocol was used to induce tumors. The individual contributions of Ha-ras and bcl-2 to papilloma latency, incidence, and growth rate in HK1.ras/bcl-2 double transgenic mice was marginally additive. Papillomas arising in HK1.ras transgenic mice exhibited the highest rate of apoptosis whereas papillomas arising in the HK1.ras/bcl-2 double transgenic mice exhibited rates of apoptosis that were significantly lower than papillomas arising in either control littermate or HK1.ras mice. Constitutive expression of either Ha-ras or bcl-2 exhibited similar rates of malignant tumor progression and they were not significantly different than control littermates. Importantly, when these two oncoproteins were coexpressed, a significant, and synergistic, increase in malignant transformation was observed.     

Trichloroethylene induce nitric oxide production and nitric oxide synthase mRNA expression in cultured normal human epidermal keratinocytes

Trichloroethylene (TCE), a major chemical hazard during occupational exposure, can cause obvious skin lesions, including irritant reactions and dermatitis. Nitric oxide (NO) synthesized by nitric oxide synthase (NOS) is involved in a broad array of pathogenesis of skin inflammatory and immune responses. To understand the mechanisms of TCE-induced dermatoxicity, we investigated the effects of TCE on NO production and NOS mRNA expression in cultured normal human epidermal keratinocytes (NHEK). Cells were treated with TCE (0 mM, 0.125 mM, 0.25 mM, 0.5 mM, 1.0 mM, 2.0 mM) for 4 h, and then incubated for 12 h, 24 h, 48 h and 72 h. At each given time point, NO production were evaluated indirectly by measuring nitrite plus nitrate concentration in the culture medium using Griess reaction, as well as cell viability determined by MTT test, iNOS and cNOS activities assayed with a NOS activity detecting kit. The expression of iNOS and cNOS mRNA was detected using RT-PCR. TCE decreases cell viability and enhance NO production from NHEK in concentration- and time-dependent manner. Aminoguanidine (AG), an inhibitor of NOS, can prevent NO production and cell viability decrease in NHEK by TCE induced. Change to NO production was accompanied by increased activities of both types of NOS, but the iNOS activity accounted mainly for the TCE-induced NO production. RT-PCR detection showed that NHEK expressed both iNOS and cNOS mRNA by TCE exposure. Whereas a concentration- and time-dependent up-regulation of the mRNA expression was observed for iNOS and cNOS following TCE exposure, changes to iNOS were more marked. These results suggest that TCE caused increase in NO production, attributed to activation of iNOS as well as cNOS, and expression of iNOS and cNOS mRNA. These cellular changes may contribute to the pathological and physiological features of TCE-induced erythema and skin inflammation.     

银屑病患者淋巴细胞对角朊细胞增殖影响的研究

用体外细胞培养技术，＾３Ｈ－ＴｄＲ标记检测培养细胞ＤＮＡ的合成，证明银屑病患者淋巴细胞对体外培养角朊细胞增殖有促进作用，与正常人淋巴细胞作用比较，Ｐ〈０．０１差异有显著性。并用地塞米松阻断银屑病患者淋巴细胞刺激表皮角朊细胞增殖，有效地建立了以银屑病患者淋巴细胞刺激表皮角朊细胞增殖的实验模型，为今后研究其他治疗药物提供了实验基础。     

Effects of recombinant human tissue inhibitor of metalloproteinases-2 (rh-TIMP-2) on migration of epidermal keratinocytes in vitro and wound healing in vivo

Tissue inhibitors of metalloproteinases (TIMP), common inhibitors of matrix proteinases, have cell-promoting activity. We studied the effects of recombinant human tissue inhibitor of metalloproteinases-2 (rh-TIMP-2) on the migration of normal human epidermal keratinocytes (NHEK). An in vitro migration assay revealed that rh-TIMP-2 enhanced random migration (up to 170%, p<0.05) in a dose-dependent manner. When we applied rh-TIMP-2 solution (20 microg/20 microl/wound) daily to full-thickness wounds made with an 8-mm punch on the backs of healthy (n=8), aged (n=9), and diabetic (n=15) rodents, we observed faster wound closure (p<0.05) than in vehicle-treated controls. Accelerated wound closure was dose-dependent (0-20 microg/wound) in diabetic mice (n=6), and the optimal concentration was 10-20 microg of rh-TIMP-2/wound. Histological examinations performed on days 0, 5, 10, 15, and 20 in diabetic mice revealed faster migration of epidermal keratinocytes from wound edges. These results suggest that rh-TIMP-2 plays an important role in wound healing.     

Intracellular calcium pool emptying induces DNA synthesis in HaCaT keratinocytes

Calcium signaling provides a central control mechanism for growth, differentiation and apoptosis of epidermal keratinocytes. Moreover, calcium signaling is important for carcinogenesis in view of the observations suggesting that emptying of intracellular stores in keratinocytes [e.g. by a selective blocker of calcium pump in the endoplasmic reticulum (ER), thapsigargin] facilitates skin cancer development. In this work, we analyzed whether calcium content in the intracellular stores is linked to HaCaT keratinocyte growth and apoptosis control. Treatment with thapsigargin caused calcium release from the intracellular pool and permanent pool depletion (up to 24 h) could be achieved using a high dose (1 micro M) of this inhibitor. HaCaT cells cultured in these conditions exhibited an increased rate of DNA synthesis, assessed by the BrdU incorporation assay. Moreover, a weak stimulation of involucrin (terminal differentiation marker) was observed. Studies where intracellular free calcium (Cai2+) was chelated with BAPTA [1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid] revealed that abrogation of thapsigargin-induced Cai2+elevation did not counteract its effects on DNA synthesis, but blocked thapsigargin-induced involucrin expression. Apoptosis was readily achieved by extracellular calcium chelation using EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], but was not observed after thapsigargin or BAPTA alone or in combination. In conclusion, depletion of intracellular calcium stores causes stimulation of keratinocyte proliferation independently of the elevation of Cai2+.     

Reciprocal altered expression of T-cadherin and P-cadherin in psoriasis vulgaris

BACKGROUND: The most characteristic change in psoriasis vulgaris is markedly increased, persistent keratinocyte proliferation. The underlying mechanism of excessive epidermal growth is controversial. We previously found and reported that T-cadherin was expressed in keratinocytes and confined to the basal layer of mouse and human skin. Invasive cutaneous squamous cell carcinoma showed a loss of T-cadherin expression. Another study showed that T-cadherin was a negative growth regulator of epidermal growth factor in T-cadherin transfectant neuroblastoma cells. OBJECTIVES: To obtain insight into the role of T-cadherin in keratinocyte proliferation and to investigate further the pathogenesis of psoriasis vulgaris, we examined the expression of T-cadherin, as well as E- and P-cadherin, in psoriasis vulgaris. METHODS: Four untreated active psoriatic skin samples from psoriasis vulgaris patients and four normal human skin samples from plastic surgery were collected, cryosectioned and immunohistochemically stained by antihuman T-, P- and E-cadherin antibodies. Further, the immunofluorescence intensities of T- and P-cadherin on the basal layer of the epidermis were quantitatively measured by the histogram function of LSM 510 software installed in a Zeiss laser scanning confocal microscope. The data were statistically analysed by Student's t-test. RESULTS: It was observed that T-cadherin was weakly and discontinuously expressed on the basal layer of psoriatic skin, while it was intensively expressed on all basal keratinocytes in normal human skin. In contrast, P-cadherin was strongly expressed throughout the entire epidermal layer in psoriatic skin samples, although its expression is restricted to the basal cell layer in normal human skin. There were no obvious differences in E-cadherin expression between normal human skin and psoriatic skin. Statistical analyses showed that the immunofluorescence intensity of T-cadherin in the basal cell layer of psoriatic skin (35 +/- 9.08) was significantly decreased compared with that in normal human skin (131.75 +/- 3.49, P = 2.46 x 10(-6)). There was a significant increase (P = 0.00139) in the immunofluorescence intensity of P-cadherin in the basal layer of psoriatic skin (68.25 +/- 12.13) compared with normal human skin (26 +/- 4.90). CONCLUSIONS: The present study demonstrates that there is downregulation of T-cadherin expression and upregulation of P-cadherin expression in psoriatic skin, which are considered to be involved in the hyperproliferation of keratinocytes in psoriasis vulgaris.     

Mast cell tryptase and chymase in chronic leg ulcers: chymase is potentially destructive to epithelium and is controlled by proteinase inhibitors

BACKGROUND: Numerous mast cells are present in chronic leg ulcers. Tryptase and chymase are the major mediators of mast cells, but their significance is mostly dependent on their activity. In addition, the proteinases may affect ulcer epithelialization. OBJECTIVES: To study levels and activity of tryptase and chymase in wash samples and biopsies from chronic leg ulcers and the possible effect of these proteinases on keratinocyte growth and adherence. METHODS: Wash samples were taken from 16 patients and a superficial shave biopsy was taken in eight of these patients; a second biopsy series was obtained from the edge of chronic venous leg ulcers (n = 6). RESULTS: Significant levels of soluble tryptase activity and histamine, but low levels of chymase activity, were measured in wash samples from chronic ulcers. No tryptase-inhibiting activity, but clear chymase-inhibiting activity, was detected in the wash samples. In superficial wound bed biopsies, relatively marked levels of chymase activity together with histamine and tryptase activity were detected. In the second biopsy series, about 80% of the mast cells belonged to the MC(TC) type (tryptase- and chymase-immunopositive). However, about 55-61% of the chymase-immunopositive cells displayed chymase activity and 64 +/- 17% of the tryptase-positive cells revealed immunoreactivity of alpha(1)-antichymotrypsin. As the activity of chymase and tryptase was detected in the ulcer base in a ratio of 1:8, a preparation containing both chymase and tryptase was partially purified from human skin yielding a similar activity ratio of 1:11-13. Treatment of fibronectin-coated plastic surfaces with this preparation decreased the adherence of cultured human keratinocytes, this effect being attributable mainly to chymase. In 2-day cultures using growth factor/serum-deficient low- or high-calcium medium, the tryptase-chymase preparation inhibited the slow growth and at higher concentrations it even induced detachment of keratinocytes. This effect was attributed to chymase, and it was partially regulated by heparin and histamine. CONCLUSIONS: Even though chymase is partially inactivated in chronic leg ulcers, accumulated mast cells in the close proximity of the epithelium edge and their chymase may impair keratinocyte adherence and migration.     

Characterization of the expression and function of N-methyl-D-aspartate receptor in keratinocytes

The N-methyl-D-aspartate (NMDA) receptor is expressed on neural tissue where it gates calcium ion entry upon stimulation. Using immunohistochemistry, it has been demonstrated in this study that the NMDAR1 receptor is also expressed on keratinocytes (KCs) in normal human skin and inflamed psoriatic skin in vivo. Furthermore, the NMDA receptor was functional as demonstrated by the ability of this receptor to trigger Ca++ influx in KCs. Incubation of cultured, human KCs with MK-801 decreases the cell growth and induces an increase in apoptosis. These findings demonstrate that the KC expression of NMDA receptor is a mechanism through which the influx of Ca++ into the cell can be regulated and suggest that the expression of this receptor may play a role in the regulation of KC growth and differentiation.