Claudin-1 expression decreases with increasing pathological grade in actinic keratosis and may be a marker of high-risk actinic keratosis.

BACKGROUND: Actinic keratosis (AK) is a common sun-induced skin disorder that can progress to invasive squamous cell carcinoma. However, there is still no reliable method to predict high-risk AK. AIM: To identify markers that reflect the biological behaviour of AK and to understand the pathogenesis of AK. METHODS: In total, 52 patients with AK and 17 site-matched healthy controls (HCs) were enrolled. We evaluated solar elastosis and immunohistochemical features using antibodies to p53, vitamin D receptor (VDR), claudin-1 and Langerin (CD207). Comparisons between AK and HC skin were performed and analyses carried out according to the pathological grade of AK. RESULTS: We found that in both patients and HCs, solar elastosis increased and Langerhans cell (LC) density decreased with ageing. Solar elastosis and p53 expression were higher and VDR expression was lower in patients than in HCs; however, there was no statistical difference between them in relation to the pathological grade of AK. Claudin-1 expression gradually decreased from HC skin to severe AK, and particularly decreased in areas with epidermal atypia. LC density in severe AK was significantly lower than in HC skin and mild AK, while there was no difference in LC density between HC skin, mild AK and moderate AK. CONCLUSIONS: Claudin-1 could be a useful marker of the pathological severity of AK. In addition, p53 increases and VDR decreases in AK, not in a gradual manner but in the early steps of carcinogenesis. LC density is relatively maintained in AK until it reaches severe dysplasia.

A case of perforating dermatofibroma with floret-like giant cells.

Dermatofibromas are slow-growing solitary nodules, composed mostly of a dermal proliferation of spindle cells and epithelioid cells. Some dermatofibromas present with multinucleated giant cells, such as Touton, foreign body, and osteoclast-like cells. We report a case of dermatofibroma containing both Touton giant cells and floret-type cells. A 12-year-old boy presented with a 6-mm, firm, nontender, dusky-red to greyish dermal nodule on his left popliteal fossa. As suggested clinically by the central opening, perforation of the epidermis with partial extrusion of the dermal components, including macrophages and vertically oriented collagen bundles, via transepidermal elimination, were detected. In the upper dermis, collagen trapping and mostly epithelioid cells with many giant cells were seen, while the lower part contained mainly spindle cells in a storiform pattern. Multinucleated giant cells scattered in the upper dermis were mainly floret-type multinucleated giant cells with star-shaped cytoplasmic projections, associated with some Touton giant cells. To our knowledge, this is the first report of a perforating dermatofibroma with floret-type multinucleated giant cells.

Role of FK506 binding proteins in neurodegenerative disorders.

Protein misfolding has been implicated in the pathophysiology of several neurodegenerative 'amyloidoses' that includes Alzheimer's, Parkinson's, Huntington's disease, frontotemporal dementia and amyotrophic lateral sclerosis. Accumulation of misfolded proteins into ordered fibrillar intra- or extracellular amyloids results in brain lesions that in turn lead to injury and neuronal loss. The appearance of protein aggregates in the diseased brain hints at an inability of cellular chaperones to properly assist folding of client proteins. Not surprisingly, studies involving cell-based and animal models of the neurodegenerative diseases have shown that overexpression of molecular chaperones can provide neuroprotection. Together with identification of new targets for symptomatic relief of motor and non-motor defects in neurodegenerative disorders, there is a critical unmet clinical need for the development of novel neuroprotective molecules. One such promising class of compounds are neuroimmunophilin ligands (NILs). Derived from FK506 (tacrolimus), NILs have been shown to be efficacious in a number of neurodegenerative disorders. The ability of these nonimmunosuppressive NILs to protect neurons is modulated, in part, by a large family of co-chaperone proteins called the FK506 binding proteins (FKBPs). This review focuses on the roles of FKBPs in neurodegenerative disorders with an emphasis on the cellular mechanisms responsible for their neuroprotective and neurotrophic activities. We discuss the structural features of FKBPs and the mode of action of NILs. For brevity, we limit our discussion to those FKBPs that are particularly enriched in the nervous system. We hope that such information will aid in the rational design of new and improved NILs for ameliorating neurodegenerative disorders.

Degradation of organic dye using zero-valent iron prepared from by-product of pickling line.

In this study, zero-valent iron (ZVI) was produced using iron oxide that is a by-product of a pickling line at a steel works. The reaction activity of the produced ZVI was evaluated through a series of decomposition experiments of Orange II aqueous solution. The size of ZVI particles increased with reduction temperature due to coalescence. Correspondingly, the specific surface area of ZVI decreased with increasing reduction temperature. The decomposition efficiency of synthesized ZVI particles was higher at a lower pH. In particular, no significant decomposition reaction was observed at pH of 4 and higher. The rate of the ZVI-assisted decomposition of Orange II was increased by addition of H2O2 at pH of 3, whereas it was reduced by addition of H2O2 at a higher pH of 6. Nevertheless, simultaneous use of ZVI, UV and H2O2 led to a considerable increase in the decomposition rate even at a high pH condition (pH = 6).

High-concentration all-trans retinoic acid induces dermal inflammation and reduces the accumulation of type I procollagen in human skin in vivo.

BACKGROUND: Because inflammation is a factor promoting ageing, all-trans retinoic acid (RA)-induced irritation may have a negative influence on collagen accumulation in human skin despite its stimulation of collagen production. OBJECTIVES: To determine whether RA-induced irritation detrimentally affects RA efficacy as represented by new collagen synthesis. METHODS: Retinoic acid (0·01%, 0·025% or 0·05%) or vehicle was applied to the buttock skin of elderly male volunteers three times a week for 8 weeks under continuous occlusion. Every 2 weeks, biopsy specimens were obtained and immunohistochemical analysis was performed to determine levels of type I procollagen expression and inflammatory cell infiltration. RESULTS: Topical RA regardless of concentration increased type I procollagen expression in human skin in vivo after 2 weeks. However, only 0·01% RA continuously increased type I procollagen expression up to 8 weeks. After 4 weeks, significant infiltrations of macrophages and neutrophils were observed in 0·025% and 0·05% RA-treated skin, and procollagen expression had returned to baseline. CONCLUSIONS: Excessive RA-induced inflammation might prevent collagen accumulation in aged skin despite the positive effect of RA on collagen production.

Deregulation of E-cadherin by human papillomavirus is not confined to high-risk, cancer-causing types.

BACKGROUND: E-cadherin is a tumour suppressor protein, which is normally expressed on keratinocytes and antigen-presenting Langerhans cells (LCs) in the epidermis. We have previously shown that E-cadherin is lost from tissues infected with the high-risk cancer-causing human papillomavirus (HPV) type 16. OBJECTIVES: To test if E-cadherin dysregulation is associated with the cancer risk of the infecting HPV and to establish if it is conserved among HPVs in the α, ß, γ and µ genera. METHODS: Forty-seven lesions infected with low- or high-risk HPV types spanning four HPV genera were stained for E-cadherin, P-cadherin and CD1a to detect LCs. RESULTS: Surface E-cadherin was reduced in tissues infected with members of the α4, α7 and α9 species and the γ and µ genera but was equivalent to normal epidermis in the ß only-infected lesions tested and patchy in α10-infected tissues. There was a direct relationship between atypical E-cadherin expression and a significant reduction in LCs. Expression of P-cadherin, a protein that is increased in the E-cadherin constitutive knockout mouse, was increased in lesions with reduced E-cadherin. CONCLUSIONS: These data show that E-cadherin dysregulation by HPV is widely conserved across the majority of HPV genera. E-cadherin expression was reduced or lost in epidermis irrespective of the cancer risk of the infecting HPV type or the ability of the virus to degrade retinoblastoma protein or p53. A correlation between dysregulated E-cadherin and reduced numbers of LCs supports viral regulation of surface E-cadherin contributing to viral evasion of the host immune system.

Slow proliferation as a biological feature of colorectal cancer metastasis.

BACKGROUND: We have recently reported an inverse relationship between colon cancer progression and tumour proliferative activity. Here, we extend our findings by evaluating the proliferative activity of liver metastatic lesions and primary colorectal cancers (CRC) that differ in their metastatic potential. METHODS: Using an earlier established multi-gene proliferation signature (GPS), proliferative levels were analysed in 73 primary CRCs and 27 liver metastases. RESULTS: Compared with primary CRCs, we observed a significantly lower expression of the GPS in liver metastases and confirmed their lower proliferative levels by quantitative RT-PCR and Ki-67 immunostaining. No difference could be detected in apoptotic indices as assessed by M30 immunostaining, indicating that the net growth rate is lower in metastases relative to primary tumours. Notably, relapsed primaries or those with established metastases had significantly lower proliferative activity than CRCs that were non-metastatic and did not relapse. CONCLUSION: Our results suggest that slow proliferation is a biological characteristic of both liver metastases and those primary tumours with the ability to metastasise. The delineation of the mechanisms underlying the inverse association between proliferation and CRC aggressiveness may be important for the development of new therapeutic strategies.

Reduced expression of a gene proliferation signature is associated with enhanced malignancy in colon cancer.

The association between cell proliferation and the malignant potential of colon cancer is not well understood. Here, we evaluated this association using a colon-specific gene proliferation signature (GPS). The GPS was derived by combining gene expression data obtained from the analysis of a cancer cell line model and a published colon crypt profile. The GPS was overexpressed in both actively cycling cells in vitro and the proliferate compartment of colon crypts. K-means clustering was used to independantly stratify two cohorts of colon tumours into two groups with high and low GPS expression. Notably, we observed a significant association between reduced GPS expression and an increased likelihood of recurrence (P < 0.05), leading to shorter disease-free survival in both cohorts. This finding was not a result of methodological bias as we verified the well-established association between breast cancer malignancy and increased proliferation, by applying our GPS to public breast cancer data. In this study, we show that reduced proliferation is a biological feature characterizing the majority of aggressive colon cancers. This contrasts with many other carcinomas such as breast cancer. Investigating the reasons underlying this unusual observation may provide important insight into the biology of colon cancer progression and putative novel therapy options.

Genetic changes in bilateral breast cancer by comparative genomic hybridisation.

The aim of this study is to find any specific genetic defect occurring frequently in bilateral breast cancer by examining the genetic changes of each chromosome using comparative genomic hybridisation (CGH). CGH was conducted for 36 breast cancer tissues taken from patients treated with surgery for bilateral breast cancer. Tumour and control DNAs were hybridised to metaphase chromosome with differential staining with fluorescein and rhodamine-dUTP. An average rate of green (DNA of tumour cell) against red (DNA of a normal peripheral blood lymphocyte) was calculated in these captured metaphase chromosomes and a ratio of more than 1.17 was defined as an acquisition, less than 0.85 as a loss and, finally, more than 2 as amplification. Twenty-six out of 36 cases (72.2%) showed a change in the number of DNA copies by CGH in one or more regions of gene. On average, 5.3 alterations for each chromosome (range, 1-14) were found, and gain was present more than loss at a ratio of 1.3:1. Loci that showed amplification were X, 17q, Xq, 8q, 14q11-21 and 17q22-qter. The locus showing the most gain was the X chromosome, which was observed in 15 (57.7%) out of 26 cases. Loss was most frequently observed in the short arm of chromosome 8. The concordance of genetic transformation of primary cancer and secondary cancer in bilateral breast cancer was an average of 18.7% in synchronous and 10.7% in metachronous cancer, showing higher similarity in synchronous breast cancer.

Phosphatidylinositol 3-kinase regulates proliferation of RAW 264.7 macrophages.

Phosphatidylinositol 3-kinase (P13-kinase) is an enzyme that acts as a direct biochemical link between a novel phosphatidylinositol pathway and a number of proteins containing intrinsic or associated kinase activities. Here we demonstrate that wortmannin, P13-kinase inhibitor, decreases the proliferation of RAW 264.7 macrophages and that another structurally unrelated inhibitor of P13-kinase, LY294002. also inhibits the proliferation. These results indicate a possible involvement of P13-kinase in RAW 264.7 macrophages growth regulation. Wortmannin stimulation of RAW 264.7 macrophages is followed by sustained expression of the mRNA of c-fos and a transient expression of the mRNA of c-jun. We also show that the wortmannin and LY294002 induce a cell cycle arrest in asynchronously growing cells leading to an inhibition of cell proliferation after 12 h of treatment. In addition, wortmannin or LY294002 inhibited the phorbol 12-myristate 13-acetate-induced macrophages proliferation potently. These results suggest that P13-kinase plays an important role in growth regulation of RAW 264.7 macrophages and that protein kinase C is a down stream effector of P13-kinase.

Transformation of kidney epithelial cells by a quinol thioether via inactivation of the tuberous sclerosis-2 tumor suppressor gene.

Although hydroquinone (HQ) is a rodent carcinogen, because of its lack of mutagenicity in standard bacterial mutagenicity assays it is generally considered a nongenotoxic carcinogen. 2,3,5-Tris-(glutathion-S-yl)HQ (TGHQ) is a potent nephrotoxic metabolite of HQ that may play an important role in HQ-mediated nephrocarcinogenicity. TGHQ mediates cell injury by generating reactive oxygen species and covalently binding to tissue macromolecules. We determined the ability of HQ and TGHQ to induce cell transformation in primary renal epithelial cells derived from the Eker rat. Eker rats possess a germline inactivation of one allele of the tuberous sclerosis-2 (Tsc-2) tumor suppressor gene that predisposes the animals to renal cell carcinoma. Treatment of primary Eker rat renal epithelial cells with HQ (25 and 50 microM) or TGHQ (100 and 300 microM) induced 2- to 4-fold and 6- to 20-fold increases in cell transformation, respectively. Subsequently, three cell lines (The QT-RRE 1, 2, and 3) were established from TGHQ-induced transformed colonies. The QT-RRE cell lines exhibited a broad range of numerical cytogenetic alterations, loss of heterozygosity at the Tsc-2 gene locus, and loss of expression of tuberin, the protein encoded by the Tsc-2 gene. Only heterozygous (Tsc-2(EK/+)) kidney epithelial cells were susceptible to transformation by HQ and TGHQ, as wild-type cells (Tsc-2(+/+)) showed no increase in transformation frequency over background levels following chemical exposure. These data indicate that TGHQ and HQ are capable of directly transforming rat renal epithelial cells and that the Tsc-2 tumor suppressor gene is an important target of TGHQ-mediated renal epithelial cell transformation.

Solution structure of the antiapoptotic protein bcl-2.

The structures of two isoforms of Bcl-2 that differ by two amino acids have been determined by NMR spectroscopy. Because wild-type Bcl-2 behaved poorly in solution, the structures were determined by using Bcl-2/Bcl-x(L) chimeras in which part of the putative unstructured loop of Bcl-2 was replaced with a shortened loop from Bcl-x(L). These chimeric proteins have a low pI compared with the wild-type protein and are soluble. The structures of the two Bcl-2 isoforms consist of 6 alpha-helices with a hydrophobic groove on the surface similar to that observed for the homologous protein, Bcl-x(L). Comparison of the Bcl-2 structures to that of Bcl-x(L) shows that although the overall fold is the same, there are differences in the structural topology and electrostatic potential of the binding groove. Although the structures of the two isoforms of Bcl-2 are virtually identical, differences were observed in the ability of the proteins to bind to a 25-residue peptide from the proapoptotic Bad protein and a 16-residue peptide from the proapoptotic Bak protein. These results suggest that there are subtle differences in the hydrophobic binding groove in Bcl-2 that may translate into differences in antiapoptotic activity for the two isoforms.

The Reliability of Histoculture Drug Response Assay (HDRA) in Chemosensitivity Tests for Breast Cancer.

PURPOSE: Cancers are highly individual in their response to chemotherapy, however attempts to predict tumor response to drugs using in vitro cell culture have largely failed. A new technology, the histoculture drug response assay (HDRA), appears to have solved many previous problems. The purpose of this study is to evaluate the reliability of HDRA in a chemosensitivity test for breast cancer. MATERIALS AND METHODS: Tumor specimens from breast cancer patients were evaluated by HDRA using different chemotherapeutic agents. Each specimen was tested using a blind method in order to determine the reproducibility of HDRA results for the same tissue and with a triplicated assay in order to determine reproducibility by different examiners. The evaluative power of this assay and the chemosensitivity of drugs for each specimen was determined. RESULTS: Specimens of 92.9% (65/70) were successfully cultured and evaluated for chemosensitivity. The reproducibility of HDRA for the same tissue was 75% (100% agreement) and 100% (over 70% agreement), respectively. And the reproducibility by different examiners was 78.9% (100% agreement) and 94.7% (over 70% agreement), respectively. Each specimen demonstrated a response to at least one agent. CONCLUSION: The evaluative power and reproducibility of HDRA were high, therefore it might serve as a reliable clinical method for chemosensitivity testing. However, there is a need for clinical trial in which patients are initially randomized for treatment either by HDRA direction or by clinician's choice.

Analysis of the conformational change of recombinant human papilloma virus type 18 E7 protein induced by metal binding.

Human papillomavirus (HPV) type 18 E7 gene was isolated by polymerase chain reaction (PCR) amplification from tissues of Korean cervical cancer patients and cloned into a plasmid vector, pET-3a, for the expression of recombinant E7 protein (rE7) in Escherichia coli. The rE7 protein was purified to the homogeneity and its purity was confirmed by HPLC. The purified protein was analyzed for the metal-binding properties by UV spectroscopy and it was shown that two Cd2+ or Zn2+ ions bind to one E7 protein by the metal-sulfur ligand formation via two Cys-X-X-Cys motifs in E7 protein. When the change of intrinsic fluorescence of tryptophan residue was analyzed for rE7-Zn complex, the blue shift of emission wavelength and the decrease in maximum intensity of emission were observed compared with rE7. These results suggest that Zn(2+)-bound rE7 has undergone conformational change, in which a tryptophan residue located in the second Cys-X-X-Cys motif was moved into solvent-inaccessible or hydrophobic environment. The rE7-Zn complex was found to be resistant to chymotrypic digestion by comparing the digestion patterns of rE7. Therefore, we showed the folding status of HPV 18 E7 could be changed by metal binding resulting in a different conformation in which a tryptophan residue was driven into more hydrophobic environment and the resistancy to chymotryptic digestion was conferred.

Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis.

Heterodimerization between members of the Bcl-2 family of proteins is a key event in the regulation of programmed cell death. The molecular basis for heterodimer formation was investigated by determination of the solution structure of a complex between the survival protein Bcl-xL and the death-promoting region of the Bcl-2-related protein Bak. The structure and binding affinities of mutant Bak peptides indicate that the Bak peptide adopts an amphipathic alpha helix that interacts with Bcl-xL through hydrophobic and electrostatic interactions. Mutations in full-length Bak that disrupt either type of interaction inhibit the ability of Bak to heterodimerize with Bcl-xL.

X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death.

THE Bcl-2 family of proteins regulate programmed cell death by an unknown mechanism. Here we describe the crystal and solution structures of a Bcl-2 family member, Bcl-xL (ref. 2). The structures consist of two central, primarily hydrophobic alpha-helices, which are surrounded by amphipathic helices. A 60-residue loop connecting helices alpha1 and alpha2 was found to be flexible and non-essential for anti-apoptotic activity. The three functionally important Bcl-2 homology regions (BH1, BH2 and BH3) are in close spatial proximity and form an elongated hydrophobic cleft that may represent the binding site for other Bcl-2 family members. The arrangement of the alpha-helices in Bcl-xL is reminiscent of the membrane translocation domain of bacterial toxins, in particular diphtheria toxin and the colicins. The structural similarity may provide a clue to the mechanism of action of the Bcl-2 family of proteins.