The Journal of Dermatolo6'} VoL It): ;;l)H-601, J9f)2
Growth and Collagen Synthesis of Cultured Neurofibroma Fibroblasts Tetsuo Sasaki, Katsuhiko Arai* and Yutaka Nagai* Abstract
Cells from cutaneous neurofibromas of three patients with von Recklinghausen's disease and skin fibroblasts from four healthy adults were cultured. After two passages, DNA and collagen syntheses were determined by measuring incorporation of [3H] thymidine and [3H] proline respectively, and expressed as the values per unit DNA content. These values from neurofibroma cells were increased by 54% in DNA synthesis and 60% (p0.05) from the control. cell density of 1 x 105 cells per well. DNA Assay Cells were cultured for 12 days. After replacing the medium with fresh solution (1 mL), the cells were incubated for two hours in the presence of 10 ,uCi of tritiated thymidine (20.0 Ci/mmol), and the medium was then removed. The cells were rinsed twice with 0.05 mol/L of TRIS hydrochloride-0.11 mol/'L of sodium chloride buffer at a pH of 7.4 and scraped into 10% chilled trichloroacetic acid. The precipitate was collected by centrifugation at 2000 g for ten minutes, washed twice with chilled trichloroacetic acid to remove free tritiated thymidine. DNA was extracted with 0.5 moUL ofperchloric acid at 70°C for 30 minutes. A1iquots of the extract were used for the assays of radioactivity and DNA content (4).
Hydroxyproline Assay Cells cultured for 11 days were incubated in fresh .medium containing 50 ,uCi of tritiated proline for 24 hours. For this purpose, commercially available L-2,3,-tritiated proline (29.1 Ci/mmol) was purified by ion-exchange chromatography using an amino acid analyzer (LCR-2 resin, 0.8 x 50 em) with 1 mollL of hydrochloric acid as an eluent as previously described (5). The combined cell layer and medium were sonicated and assayed as follows. An aliquot of the sonicated material was directly lyophilized without dialysis, hydrolyzed with 0.5 mL of 6 mol/L of hydrochloric acid in an evacuated sealed tube at 110°C for 24 hours, and dried. The resulting hydrolyzate was dissolved in citrate buffer (0.2 mol/L of Na") at a pH of 2.2, filtered through
Cell
IntraTotal Nondialyzable hydroxyhydroxycellular proline proline degradadpmlng dpmlng tion a of DNA of DNA %
Control 1 131 184 2 3 166 4 292 Mean ± SD 193±61 (n=4) Patient 1 355 364 2 3 222 Mean ± SD 314±65 (1.63)b,c (n=3)
98 121 118 169 127±26
24.6 34.2 28.7 42.2 32.4±6.6
221 230 157 203±33 (1.60)b,d
37.6 36.8 29.6 c 34.7±3.6
Expressed as percent of dialyzable hydroxyproline per total hydroxyproline. bThe value in the parentheses is the ratio relative to the control. cNot significantly different (p>0.05) from the control. dSignificantly different (p
Growth and Collagen Synthesis of Cultured Neurofibroma Fibroblasts Tetsuo Sasaki, Katsuhiko Arai* and Yutaka Nagai* Abstract
Cells from cutaneous neurofibromas of three patients with von Recklinghausen's disease and skin fibroblasts from four healthy adults were cultured. After two passages, DNA and collagen syntheses were determined by measuring incorporation of [3H] thymidine and [3H] proline respectively, and expressed as the values per unit DNA content. These values from neurofibroma cells were increased by 54% in DNA synthesis and 60% (p0.05) from the control. cell density of 1 x 105 cells per well. DNA Assay Cells were cultured for 12 days. After replacing the medium with fresh solution (1 mL), the cells were incubated for two hours in the presence of 10 ,uCi of tritiated thymidine (20.0 Ci/mmol), and the medium was then removed. The cells were rinsed twice with 0.05 mol/L of TRIS hydrochloride-0.11 mol/'L of sodium chloride buffer at a pH of 7.4 and scraped into 10% chilled trichloroacetic acid. The precipitate was collected by centrifugation at 2000 g for ten minutes, washed twice with chilled trichloroacetic acid to remove free tritiated thymidine. DNA was extracted with 0.5 moUL ofperchloric acid at 70°C for 30 minutes. A1iquots of the extract were used for the assays of radioactivity and DNA content (4).
Hydroxyproline Assay Cells cultured for 11 days were incubated in fresh .medium containing 50 ,uCi of tritiated proline for 24 hours. For this purpose, commercially available L-2,3,-tritiated proline (29.1 Ci/mmol) was purified by ion-exchange chromatography using an amino acid analyzer (LCR-2 resin, 0.8 x 50 em) with 1 mollL of hydrochloric acid as an eluent as previously described (5). The combined cell layer and medium were sonicated and assayed as follows. An aliquot of the sonicated material was directly lyophilized without dialysis, hydrolyzed with 0.5 mL of 6 mol/L of hydrochloric acid in an evacuated sealed tube at 110°C for 24 hours, and dried. The resulting hydrolyzate was dissolved in citrate buffer (0.2 mol/L of Na") at a pH of 2.2, filtered through
Cell
IntraTotal Nondialyzable hydroxyhydroxycellular proline proline degradadpmlng dpmlng tion a of DNA of DNA %
Control 1 131 184 2 3 166 4 292 Mean ± SD 193±61 (n=4) Patient 1 355 364 2 3 222 Mean ± SD 314±65 (1.63)b,c (n=3)
98 121 118 169 127±26
24.6 34.2 28.7 42.2 32.4±6.6
221 230 157 203±33 (1.60)b,d
37.6 36.8 29.6 c 34.7±3.6
Expressed as percent of dialyzable hydroxyproline per total hydroxyproline. bThe value in the parentheses is the ratio relative to the control. cNot significantly different (p>0.05) from the control. dSignificantly different (p
