1. Adenosine- and Adenine-Nucleotide-Mediated Inhibition of Normal and Transformed Keratinocyte Proliferation is Dependent upon Dipyridamole-Sensitive Adenosine Transport 
Jeffrey R. Brown, Paul W. Cook 
Journal of Investigative Dermatology 
Volume 115, Issue 5, Pages (November 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Human keratinocytes express A2B purinoreceptor mRNA. Human keratinocytes were cultured and subjected to northern blot analysis to detect mRNA corresponding to the human A2B adenosine receptor, as described in Materials and Methods. Data are representative of the results from three different strains of normal human keratinocytes.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Human keratinocytes display adenosine-, AMP- and adenosine- receptor-agonist-dependent elevation of intracellular cAMP. Human keratinocytes were cultured and assayed for intracellular cAMP as described in Materials and Methods. (A)-(D) 200 μM theophylline (Theo) was added prior to the addition of other experimental agents as indicated in the figure. Each data point is plotted as the percentage of maximal cAMP level attained by treatment with forskolin (Forsk, 50 μM), and represents the average of two determinations ± the range.
Journal of Investigative Dermatology  , DOI: ( /j x)
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4. Figure 3
Human keratinocytes express hENT1-specific mRNA. Human keratinocytes were cultured and subjected to northern blot analysis to detect mRNA corresponding to hENT1, as described in Materials and Methods. Data are representative of the results from three different strains of normal human keratinocytes.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Human keratinocytes display dipyridamole-sensitive adenosine transport. Human keratinocytes were cultured and assayed for adenosine transport as described in Materials and Methods. (A) Cells were treated with 0.17 μM [2,8-3H]adenosine in the presence or absence of unlabeled nucleosides and nucleotides (170 μM) or unlabeled nucleoside transport blocking agents (17 μM). Bars represent the average percentage maximal inhibition of [2,8-3H]adenosine incorporation. (B), (C) Cells were treated with 0.1 μM [2,8-3H]adenosine in the presence or absence of unlabeled nucleosides and nucleotides at the indicated concentrations. Percentage inhibition of [2,8-3H]adenosine uptake was measured as described in Materials and Methods and for (A). Each point is the average determination from three different cell wells, ± the standard error of the mean (SEM).
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
Nucleoside transport blocking agents completely reverse the antiproliferative activity of adenosine on human keratinocytes. Human neonatal keratinocytes were cultured as described under ‘‘Clonal growth assays’' in Materials and Methods. (A)-(D) ADO was added to the keratinocyte cultures to produce a final concentration of 50 μM. Adenosine-treated cells were cultured in the presence or absence of the nucleoside transport blocking agents dipyridamole, NBTI, dilazep, and lidoflazine, at the indicated concentrations. After subsequent culture for 9 d, the cells were fixed, stained, and imaged as described in Materials and Methods. Each well is representative of three colony growth determinations.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
Nucleoside transport blocking agents completely reverse the antiproliferative activity of adenine nucleotides on human keratinocytes. Human neonatal keratinocytes were cultured as described under ‘‘Clonal growth assays’' in Materials and Methods. (A)-(C) Adenine nucleotides (AMP, ADP, ATP) were added to the keratinocyte cultures to produce a final concentration of 50 μM. Adenine-nucleotide-treated cells were cultured in the presence or absence of nucleoside transport blocking agents (dipyridamole, NBTI, dilazep), at the indicated concentrations. After subsequent culture for 9 d, the cells were fixed, stained, and imaged as described in Materials and Methods. Each well is representative of three colony growth determinations.
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
ADA abrogates the antiproliferative activity of adenosine, but not adenine nucleotides, on human keratinocytes. Human neonatal keratinocytes were cultured as described under ‘‘Clonal growth assays’' in Materials and Methods. (A) ADO, AMP, ADP, or ATP was added to the keratinocyte cultures to produce a final concentration as indicated in the figure. (B) As (A) except that ADA was added at a concentration of 2 U per ml. After subsequent culture for 9 d, the cells were fixed, stained, and imaged as described in Materials and Methods. Each well is representative of three colony growth determinations.
Journal of Investigative Dermatology  , DOI: ( /j x)
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9. Figure 8
Adenosine and adenine nucleotides inhibit the proliferation of transformed epidermal cells. The transformed epidermal cell line SCC-25 was cultured for 24 h as described under ‘‘Clonal growth assays’' in Materials and Methods. (A) ADO, AMP, ADP, or ATP was added to the SCC-25 cells to produce a final concentration as indicated in the figure. (B) 50 μM ADO or AMP was added to the SCC-25 cells in the presence or absence of 300 nM dipyridamole, as indicated in the figure. After subsequent culture for 9 d, the cells were fixed, stained, and imaged as described in Materials and Methods. Each well is representative of three colony growth determinations.
Journal of Investigative Dermatology  , DOI: ( /j x)
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10. Figure 9
A murine keratinocyte-like cell line can be made resistant to the antiproliferative effects of adenosine and adenine nucleotides. BALB MK-2 cells and the derived adenosine-resistant population were cultured as described in the text and under ‘‘Clonal growth assays’' in Materials and Methods. (A) ADO, AMP, ADP, or ATP was added to the BALB MK-2 cells to produce a final concentration as indicated in the figure. (B) ADO, AMP, ADP, or ATP was added to the adenosine-resistant BALB MK-2 cells to produce a final concentration as indicated in the figure. After subsequent culture for 9 d, the cells were fixed, stained, and imaged as described in Materials and Methods. Each well is representative of three colony growth determinations.
Journal of Investigative Dermatology  , DOI: ( /j x)
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11. Figure 10
Adenosine and AMP arrest the proliferation of human keratinocyte cultures within 12–24 h. Human neonatal keratinocytes were cultured as described under ‘‘Human keratinocyte high-density proliferation assay’' in Materials and Methods. After 24 h of culture, 300 μM adenosine, AMP, or no nucleoside/tide (Control) was added to the cell cultures for the designated period of time, as indicated in the figure. All cultures received 0.1% DMSO. Each point represents the average cell number from three different cell wells, ± the standard error of the mean (SEM).
Journal of Investigative Dermatology  , DOI: ( /j x)
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12. Figure 11
Adenosine and adenine nucleotide treatment of human keratinocyte cultures decreases the percentage of G2-phase cells and increases the percentage of S-phase cells. Forty-eight hours prior to dissociation and cell cycle analysis, 300 μM ADO, AMP, ADP, or ATP was added to the keratinocyte cultures. Alternatively 0.1 μM demicolcine was added 24 h prior to dissociation and cell cycle analysis. Keratinocytes were subjected to DNA staining and flow cytometry as described in Materials and Methods. (A)-(E) Flow cytometry profiles of ADO-, AMP-, ADP-, ATP- or demicolcine-treated keratinocytes, respectively. Each profile represents a response that is representative of three independent determinations.
Journal of Investigative Dermatology  , DOI: ( /j x)
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13. Figure 12
Models of human keratinocyte ectonucleotidase-adenosine transport complex in human keratinocytes. Two hypothetical models of a cell surface ectonucleotidase-adenosine transport system in human keratinocytes.
Journal of Investigative Dermatology  , DOI: ( /j x)
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