Differential degradation of [35S]methionine polypeptides in Duchenne muscular dystrophy skin fibroblasts in vitro.

Abstract

Rates of protein turnover have been measured in three normal and three Duchenne muscular dystrophy (DMD) skin fibroblast cell lines. Cell populations were analyzed at identical states with regard to cell number, state of topoinhibition, and cumulative population doublings (CPD). Net protein synthesis measured by the incorporation of [35SSmethionine in an 18-hr pulse was reduced by an average of 34%; degradation of total cellular protein measured after an 18-hr pulse with [35S]methionine and a 24-hr chase was enhanced by an average of 50% in DMD fibroblasts. Twodimensional gel electrophoresis analyses revealed that the breakdown of the majority of [35S]methionine polypeptides was markedly increased in DMD fibroblasts. Quantitative determinations of the differential degradation rates of 10 selected proteins in the tropomyosin region of two-dimensional gels were undertaken by scintillation counting and computer analyses. In three series of experiments, the degradation of the 10 proteins in DMD fibroblasts was enhanced by individual polypeptides between 12.0% and 151.2% as measured by scintillation counting or between 0.8% and 128% as determined by computer analyses.Duchenne muscular dystrophy (DMD) is an X-chromosomelinked recessive disorder, characterized by a progressive muscle degeneration in the primary organs of manifestation, the skeletal and cardiac muscles. The primary genetic defect of this disorder has not been defined; dystrophy of skeletal and cardiac muscles is considered to be the result of an abnormal protein metabolism (1-3). Analyses of the primary and/or secondary biochemical defects in degenerating muscle in DMD in vivo have been hampered by secondary changes-e.g., concomitant regeneration and degeneration of myoblasts, fibroblasts, and fat cells (1). The secondary changes make the selection of an appropriate reference base and of a representative control in vivo difficult.Numerous cellular and biochemical abnormalities have also been demonstrated in nerve, erythrocyte, and fibroblast cells in DMD patients (1, 4, 5). DMD skin fibroblast populations in vitro express a variety of syndrome-specific cellbiological (6-10), and biochemical (11-17) (20).In the present study, we analyzed net protein synthesis and degradation of total cellular protein and of individual proteins in DMD and normal skin fibroblasts by two-dimensional gel electrophoresis. We will provide evidence that in DMD skin fibroblasts the majority of proteins are affected by a decreased net synthesis and an enhanced degradation. Analyses ofthe degradation of individual [35S]methionine polypeptides in the tropomyosin region of two-dimensional gels revealed a significant polypeptide-specific increase in the rates of degradation in DMD fibroblasts. MATERIALS AND METHODSCell Cultures. Skin fibroblast populations of three male patients (6-15 yr old) with advanced to very advanced manifestations of the DMD syndrome and of three agematched normal male probands were established from skin biopsies as described elsewhere (14). Three DMD ...

Abnormal Growth and Clonal Proliferation of Fibroblasts Derived from Kidneys with Interstitial Fibrosis

“…Pure renal fibroblast clones (CTF I and CTF 11) of NKF and FKIF cells selected as described above were labeled with 50 pCi of [35S]methionine in L-methioninefree medium (DMEM) supplemented with 1 mg/liter of L-methionine and 20% FCS for 18 hr as described elsewhere (6,8,10). After labeling, radioactive medium was removed and centrifuged to remove cells and cellular debris.…”

“…After labeling, radioactive medium was removed and centrifuged to remove cells and cellular debris. Medium was lyophylized in 200-pl samples and reconstituted in 50 p1 of modified O'Farrell lysis buffer (6,8,10). Clonal cell layers were washed three times with phosphate-buffered saline and lysed in 100 pl of buffer (6).…”

Human skin fibroblasts in vitro differentiate along a terminal cell lineage.

“…Primary skin fibroblast populations of the cell line HH-8 were established from the lower right abdominal region of an 8-yr-old female donor as described (17 (18). After labeling, homogeneous subpopulations (50-100 cells) of mitotic fibroblasts F I, F II, and F III and of postmitotic fibroblasts F IV, F V, and F VI were surrounded by silicone-sealed glass rings.…”

“…After labeling, homogeneous subpopulations (50-100 cells) of mitotic fibroblasts F I, F II, and F III and of postmitotic fibroblasts F IV, F V, and F VI were surrounded by silicone-sealed glass rings. Cells were lysed by lysis buffer, and after determination ofradioactivity, aliquots of the various samples (containing 500,000 cpm) were analyzed by two-dimensional polyacrylamide gel electrophoresis, as described (18). (Fig.…”

Differential degradation of intracellular proteins in human skin fibroblasts of mitotic and mitomycin-C (MMC)-induced postmitotic differentiation states in vitro

“…Human skin fibroblasts of the cell line HH-8, established from the lower abdominal skin of an 8-year-old female donor [3] were maintained and subcultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (Boehringer, Mannheim, lot No. 881) and standard amounts of antibiotics as previously described [3,27,291. Growth rates and cumulative population doublings (CPD) were determined by counting the cells at each passage in a Fuchs-Rosenthal hemocytometer.…”