A comparison of the changing patterns of crystallin expression in vivo, in long-term primary cultures in vitro and in response to a carcinogen.

Changes in the differentiation of day-old chick lens epithelium in long-term primary culture conditions were investigated by sodium dodecyl sulphate-polyacrylamide electrophoresis, using integrating densitometry to assess the relative levels of accumulated crystallin and non-crystallin polypeptides and fluorography to assess their relative levels of synthesis. The main changes during the culture period included a relative decline in the proportion of actin and other non-crystallins, an initial increase in 48K delta-crystallin expression followed by a decline and a shift in beta-crystallin expression from a relative preponderance of the 24K and 23K polypeptides to a relative preponderance of the 24K and 22K polypeptides. At all stages the level of the 19K alpha-crystallin was higher than that of the 20K alpha-crystallin polypeptide. In general, the changes in the pattern of expression of these polypeptides in culture were similar to those observed in vivo in the post-hatch chick, suggesting an intrinsic programme of crystallin expression. The changes in gene expression were also tested indirectly by brief exposure of the cells in vitro to a carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) which is known to produce, in some systems, effects related to the status of the cell at the time of treatment. The effects were found to depend on the stage of differentiation of the culture at the time of treatment. Treatment on day 1 of culture prevented later lentoid formation and severely reduced the expression of all crystallins with the exception of the 34K beta-crystallin polypeptide. Actin was the most abundant soluble cell component, and a proportion of the cells acquired a fibroblast-like morphology. Treatment with MNNG on day 7 led to a delay in lentoid formation and a differential reduction of the synthesis of crystallin polypeptides, whereas the treatment of already differentiated cultures on day 18 and to lesser extents on days 27, 45 and 55, respectively, led to an increase in crystallin synthesis relative to controls. These results suggest that this programme of crystallin gene expression becomes more resistant to change with increasing epithelial differentiation.