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Volumn 285, Issue 5429, 1999, Pages 898-900

Fas ligand: A sensor for DNA damage critical in skin cancer etiology

Author keywords

[No Author keywords available]

Indexed keywords

FAS ANTIGEN; FAS LIGAND; PROTEIN P53;

EID: 0033529581     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5429.898     Document Type: Article
Times cited : (229)

References (28)
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    • A. Ziegler et al., Nature 372, 773 (1994).
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    • S. Nagata, Cell 88, 355 (1997).
    • (1997) Cell , vol.88 , pp. 355
    • Nagata, S.1
  • 12
    • 0345003056 scopus 로고    scopus 로고
    • note
    • 2) corresponding to 2.3 minimal erythemal doses for C3H mice.
  • 13
    • 0344572048 scopus 로고    scopus 로고
    • note
    • Formaldehyde-fixed skin biopsies were paraffin-embedded, and immunohistochemistry was performed as previously described (27) with polydonal goat antibody to Fas (X-20), FasL (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA), or normal goat serum (isotype control) at final dilutions of 1:500. Staining was visualized with stable diaminobenzidine (Research Genetics, Huntsville, AL). Specificity of staining was confirmed with blocking peptides; preincubation of antibody with specific peptide eliminated all staining. FasL staining was also verified with a monoclonal antibody to FasL (KAY-10; Pharmingen).
  • 14
    • 0344572046 scopus 로고    scopus 로고
    • note
    • Shaved dorsal skins were fixed in buffered 10% formalin and embedded in paraffin. Five-micrometer sections were stained with hematoxytin-eosin and examined by light microscopy. Criteria for scoring sunburn cells were intensely staining eosinophilic cytoplasm, pycnotic nuclei, and separation from adjacent cells as previously described (2). For each of two to five mice per point, 10 hematoxytin-eosin sections from two separate epidermal sections were counted for sunburn cells. Sunburn celt incidence was calculated per linear centimeter of epidermis with coded slides that did not reveal treatment group. No detectable differences between wild-type and gld/gld mice in morphology, organization, or thickness were observed in the epidermis.
  • 15
    • 0344140567 scopus 로고    scopus 로고
    • Dorsal skins were fixed in buffered 10% formalin and embedded in paraffin. TUNEL assay was done with a commercially available kit according to the manufacturer's directions (Promega, Madison, WI). Slides were examined with an Olympus Inverted System Microscope 1X70 (Melville, NY), and pictures were taken with a Nikon 35-mm camera (magnification, X 10)
    • Dorsal skins were fixed in buffered 10% formalin and embedded in paraffin. TUNEL assay was done with a commercially available kit according to the manufacturer's directions (Promega, Madison, WI). Slides were examined with an Olympus Inverted System Microscope 1X70 (Melville, NY), and pictures were taken with a Nikon 35-mm camera (magnification, X 10).
  • 16
    • 0344140566 scopus 로고    scopus 로고
    • L. L. Hill et al., data not shown
    • L. L. Hill et al., data not shown.
  • 17
    • 0344572044 scopus 로고    scopus 로고
    • Data can be accessed at www.sciencemag.org/ feature/data/1040041.shl
    • Data can be accessed at www.sciencemag.org/ feature/data/1040041.shl
  • 18
    • 0345434177 scopus 로고    scopus 로고
    • L. L. Hill and L. B. Owen-Schaub, unpublished observations
    • L. L. Hill and L. B. Owen-Schaub, unpublished observations.
  • 22
    • 0345003053 scopus 로고    scopus 로고
    • note
    • 2 pieces of dorsal skin from the midline of each mouse was separated from the dermis by floating the skin dermis side down in phosphate-buffered saline containing 0.25% trypsin for 2 hours at 37°C followed by mechanical separation and freezing of epidermis. Frozen epidermis was digested in lysis buffer [4 M urea, 0.5% N-lauroylsarcosine, 10 mM EDTA, 0.2 M NaCl, and 100 mM tris-HCI (pH 8.0)] containing proteinase K (300 μg/ ml) and ribonuclease (100 μg/ml) at 37°C overnight. Samples were heated at 60° for 15 min, and the DNA was extracted with the phenol-chloroform method. The primer sequences used were as follows: codon 270, 5′-GGACGGGACAGCT TTGAGGTTT-3′ (forward); codon 275, 5′-GTGTTTGTGCCTGCCT-3′ (forward); and 3′ prime end of exon 8, 5′-GCCTGCGTACCTCTCTTTGC-3′. The PCR product was resolved on a 6% polyacrylamide gel and visualized on autoradiographic film.
  • 28
    • 0345003051 scopus 로고    scopus 로고
    • We thank L. Eichler for expert technical assistance and M. Duvic and D. McConkey for critical review of this manuscript. This work was supported by grants from the Skin Cancer Foundation, American Cancer Society (CIM-96070 to L.O.-S.), and NIH (CA45623 to H.N.A. and CA52457 to L.O-S.). LLH. is the recipient of the Crockrell University Cancer Fighters Scientific Achievement Award and an NIH postdoctoral fellowship (F32 AI09351)
    • We thank L. Eichler for expert technical assistance and M. Duvic and D. McConkey for critical review of this manuscript. This work was supported by grants from the Skin Cancer Foundation, American Cancer Society (CIM-96070 to L.O.-S.), and NIH (CA45623 to H.N.A. and CA52457 to L.O-S.). LLH. is the recipient of the Crockrell University Cancer Fighters Scientific Achievement Award and an NIH postdoctoral fellowship (F32 AI09351).


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