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

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Abstract
Summary: The 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; nDMD = 5; ncontrols = 4) whereas plating efficiency is equal for both DMD and controls. On day 7 the activity of the membrane bound enzyme 5‘-nucleotidase either per mg protein or per μg DNA is significantly elevated in cells from DMD patients (P < 0.0005; nDMD = 8; ncontrols = 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 from DMD fetuses, they could serve as a potential prenatal diagnosis of the disease. Speculation: As a systemic disease, Duchenne muscular dystrophy is also expressed in cultured skin fibroblasts by a growth delay of so far unknown origin. Since cultured amniotic cells are metabolically similar to fibroblasts, corresponding growth kinetic alterations might be expected in amniotic cells from DMD fetuses. This would provide a suitable method for prenatal diagnosis.