Differentiation Effect Of Pyruvate And Uridine On Cultured U937-ρ°cells

Background & ObjectiveLong-term exposure of cells to an appropriate concentration of ethidium bromide inhibits mitochondrial DNA(mtDNA) replication and transcription,but has no effect on the replication of nuclear DNA.This has been reported to result in cellular subclones depleted of mitochondrial DNA(mtDNA).Such mtDNA-depleted(ρ°) subclones contain disorganized and disformed mitochondria and are auxotrophic for uridine and pyruvate.Establishingρ°cell lines is of importance for studies of the mechanisms of interactions between mtDNA and nuclear genome,including the roles of mtDNA molecules integrated in the nuclear genome.These cell lines are also of importance for studies of possible roles of mitochondria or mtDNA molecules in apoptotic process.Using U937 cells and U937-ρ°cells,Marchetti et al have shown that both cells undergo the same early apoptosis-associated changes induced by tumor necrosis factor-αand cycloheximide.The human pro-monocytic leukemia U937 cell line was previously reported to becomeρ°cells after a long term ethidium bromide exposure.In our extensive PCR studies with different pairs of primers for the mtDNA molecule we showed that these U937-ρ°cells,after being cultured in our laboratory for a period,did replete their mtDNA.That the cells grew well in the normal medium(RPMI 1640 plus 10%fetal calf serum and 2 mM L-glutamine) as the parental cells also suggests that these cells contain functional mitochondria and mtDNA molecules.Further experiments showed that the cells cultured in the medium with pyruvate and uridine rather rapidly and strongly adhered on the culture flask walls while the cells cultured in the medium without pyruvate and uridine either floated or only very loosely covered the culture flask walls. Ultrastructural examination showed that the floating cells,which were often found in the culture without pyruvate and uridine,were rather similar to monocytes in nature, like the original U937 cells,while the attached larger cells appearing earlier in the culture with pyruvate and uridine demonstrated macrophage differentiation,indicating a differentiation effect of pyruvate and uridine on the cells.In our present report,mtDNA repletion and the influence of pyruvate and uridine on cell attachment were studied based on the U937-ρ°cells.The mtDNA intact status of the U937-ρ°cell line was extensively studied,and then the effect of pyruvate and uridine on cell attachment was ultrastructurally analyzed.We found that the U937-ρ°cells somehow could recover functional mitochondria and intact mtDNA molecules, and pyruvate and uridine exerted differentiation effects on these cellsMethodsComparative cell culture of the U937-ρ°cells.The cells were equally divided and comparatively cultured in the medium with either the sodium pyruvate and uridine as one group,or without the sodium pyruvate and uridine as another group.The morphological features of both cell cultures were photographically documented every day.Electron microscopyThe cells were collected and processed for electron microscopy by applying a conventional method.PCR analysisThe mtDNA was amplified by PCR using 4 mtDNA primer pairs overlapping the mitochondrial DNA.ResultsComparative cell culture of the U937-ρ°cells.The cells grew well and could adhere to the wall of the culture flasks quickly.in the medium containing sodium pyruvate and uridine,while the cells cultured in the medium without pyruvate and uridine,could form spherical colonies which floated in the medium or loosely attached on the flask.Electron microscopyThe attached cells were larger in size than the floating cells.Both cells demonstrated a number of bizarre and prominent nucleoli.The larger cells were rich in organelles,such as mitochondria,rough endoplasmic reticulum,Golgi complexes,ribosomes, multivesticular bodies and coated vesicles.Especially,these cells were rich in lysosomes,both primary and secondary,with long cytoplasmic processes and curved or invaginated nuclear membranes,indicating macrophage differentiation.The small cells within spherical colonies or floating cells contained fewer organelles,shorter and mainly rounded cytoplasmic processes.Mitochondria in both groups of cells displayed no special defects.They were rich in cristae with a compact matrix.PCR resultsAll the primer pairs gave satisfactory PCR products under optimised conditions.ConclusionThe cultured cell could adhered on the culture flask walls or not indicating the differentiation effect of pyruvate and uridine on the cells.U937-ρ°cells could recover functional mitochondria and intact mtDNA molecule.