Effect of confluence on endocytosis by 3T3 fibroblasts: increased rate of pinocytosis and accumulation of residual bodies

We have compared lysosomal enzyme distributions on density gradients and rates of transport of endocytic markers for actively-growing and confluent cells. While it has been previously established that mammalian cells accumulate lysosomal enzymes during quiescence, we show that this accumulation is predominantly in residual bodies (p greater than 1.12 g/ml) rather than in dense lysosomes (p = 1.08-1.10 g/ml) and does not represent a change in the endosomal and lysosomal enzyme content. The accumulation is not caused by a change in the rate of production of dense lysosomes, since the rate of transfer of epidermal growth factor (EGF) from light to dense compartments is the same between confluent and subconfluent cells. Confluent cultures have a higher rate of initial pinocytosis, and a higher rate of retroendocytosis and/or recycling, causing a net lower rate of accumulation of fluid-phase material. The accumulation of residual bodies in confluent cultures may be caused by a lower rate of exocytosis of their contents and/or a lack of dilution by cell division. The data indicate that the impact of culture confluence must be carefully assessed in experiments designed to analyze endocytic pathways.     

Regulation of transforming growth factor alpha and transforming growth factor beta messenger ribonucleic acid abundance in T-47D, human breast cancer cells

Both transforming growth factor beta (TGF beta) and TGF alpha mRNA are expressed in human breast cancer cell lines. We have investigated the relationship of mRNA abundance for these growth modulators to the proliferation rate of a number of human breast cancer cell lines. Furthermore, we have investigated the relationship of regulation of TGF beta and TGF alpha mRNA to growth inhibition caused by progestins and nonsteroidal antiestrogens in T-47D human breast cancer cells. The abundance of TGF beta and TGF alpha mRNA in human breast cancer cell lines was not related directly to proliferation rate of the cells in culture or estrogen receptor positivity or negativity. The relationship of TGF beta and TGF alpha mRNA to growth inhibition caused by antiestrogens and progestins was investigated in T-47D human breast cancer cells. We observed that in T-47D human breast cancer cells the abundance of TGF beta mRNA is decreased in a time- and dose-dependent fashion by progestins but remains unaltered by nonsteroidal antiestrogens. Treatment of T-47D cells for 24 h with 10 nM medroxyprogesterone acetate (MPA) reduced the level of TGF beta mRNA to one third that present in untreated cells. The same treatment increased TGF alpha mRNA 3-fold above untreated controls in a time- and dose-dependent fashion and nonsteroidal antiestrogens caused a small decrease. The regulation of both TGF alpha and TGF beta mRNA was not directly related to inhibition of growth by progestins and antiestrogens in T-47D cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the erythrocyte superoxide dismutase allozymes in the deer Cervus elaphus

The cDNA sequences for Cu,Zn superoxide dismutase from two Cervus elaphus subspecies, North American wapiti and European red deer, were determined. The derived amino acid sequences showed two differences: residue 8 was Leu in wapiti and Met in red deer and residue 25 was His in wapiti and Asn in red deer. The extra positive charge at position 25 in the wapiti isoform accounted for its greater mobility towards the cathode during non-denaturing electrophoresis, a procedure widely used in the genetic analysis of deer. There was no difference in specific activity between the two Cu,Zn superoxide dismutase isoforms, but the wapiti isoform was slightly more susceptible to heat denaturation.