Abnormal Growth Kinetics and 5′-Nucleotidase Activities in Cultured Skin Fibroblasts from Patients with Duchenne Muscular Dystrophy

Abstract

SummaryThe experiments reported herein compare growth kinetics and biochemical properties of cultured skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) and matched normal controls.On day 7 after plating (6000 cells/cm2) cell number and DNA per dish are significantly reduced ( P < 0.001) in the cultures from DMD patients (n = 14)' compared to those from controls (n = 10). Moreover DMD cells contain less lipids and proteins per dish but more per cell than normal fibroblasts (not significant). Variations of media (McCoy's medium instead of Eagle's minimum essential medium) and sera (human cord serum instead of fetal calf serum) resulted in the same differences between DMD and control cells.Cell kinetic experiments (plating density: 2000 cells/cm2) show increased doubling times of DMD fibroblasts ( P < 0.001; ~D M D = 5; n,o,t,o~s = 4) whereas plating efficiency is equal for both DMD and controls.On day 7 the activity of the membrane bound enzyme 9-nucleotidase either per mg protein or per pg DNA is significantly elevated in cells from DMD patients ( P < 0.0005; nDMD = 8; nCont,,ls = 9) independent of cell density. Thus all findings in cultured DMD fibroblasts: increased doubling time, tendency to more voluminous cells, and elevated 5'-nucleotidase activity per cell suggest, that the DMD cells behave similar to prematurely aging cells. Until now we were not able to check whether any alterations of the plasma membrane are inducing early senescence or, reversely, premature aging is the cause of the postulated membrane alterations.If these findings were to be confirmed in cultured amniotic cells 13, 27, 40). All these findings are supposed to be secondary or tertiary alterations. The underlying metabolic defect is not yet known. Cultured fibroblasts obtained from explants of human skin biopsies have proved to be very useful for the investigations of numerous inborn errors of metabolism. Studies of cultured fibroblasts from DMD patients have been initiated only very recently. There are reports of altered morphology (43), reduced cell aggregation (17), decreased protein synthesis (5, 29), and decreased lysosomal dipeptidyl aminopeptidase I activity (1 1).Herein we report the results of investigations on cultured fibroblasts from patients with DMD: plating efficiency, doubling time, effects of different media and sera, 14C-thymidine incorporation in DNA, and activity of the membrane bound enzyme 5'-nucleotidase (EC 3.1.3.5.). MATERIALS AND METHODS MATERIALWe studied fibroblasts growing from skin biopsies of 14 patients with DMD, aged 4 to 14 yr, and of 10 normal boys matched by age, who were undergoing surgical treatment. The cells were usually cultured in Eagle's minimum essential medium (MEM), supplemented with nonessential amino acids and 10% fetal calf serum (FCS). To test the influence of different growth conditions, McCoy's 5A modified medium (26) was used instead of MEM, and MEM was supplemented with 10% human cord serum instead of FCS. METHODS Dfrom DMD fetuses, they could serve as a potential ...

Letter to the Editor: Premature Aging of Fibroblasts from Patients with Duchenne Muscular Dystrophy

“…The results presented by Liechti-Gallati, et al (4) appear identical to data obtained when early and late passage cultures are compared (3). The most striking differences reported for DMD fibroblasts were diminished doubling frequency during log phase growth and increased 5' nucleotidase activity (4).…”

“…A recent publication by Liechti-Gallati, et al, entitled "Abnormal Growth Kinetics and 5' Nucleotidase Activities in Cultured Skin Fibroblasts from Patients with Ducheme Muscular Dystrophy" claimed that these fibroblasts have altered growth characteristics which represent a premature aging syndrome (4). Their data is in direct conflict with our published findings that show no alterations in the growth kinetics or the in vitro lifespan of fibroblasts from patients with Duchenne muscular dystrophy (9,10).…”

“…Their data is in direct conflict with our published findings that show no alterations in the growth kinetics or the in vitro lifespan of fibroblasts from patients with Duchenne muscular dystrophy (9,10). Ducheme muscular dystrophy (DMD) is an X-linked recessive disorder (4), with no clinical indications of premature aging. For this disorder to be a segmental progeroid syndrome, there should consistently be a diminished in vitro replicative lifespan, as is the case with progeria and Werner's syndrome (5).…”

“…For this disorder to be a segmental progeroid syndrome, there should consistently be a diminished in vitro replicative lifespan, as is the case with progeria and Werner's syndrome (5). Thus the explanation for the differences in our two sets of data suggests that the diminished response reported by Liechti-Gallati, et al (4) may not be an intrinsic property of DMD fibroblasts.…”

“…The most striking differences reported for DMD fibroblasts were diminished doubling frequency during log phase growth and increased 5' nucleotidase activity (4). A delay in log phase doubling time is a common feature of nearly senescent cultures (3).…”