A human diploid fibroblast cell line has been established from the lung tissue of a male fetus. This has been characterized and frozen away in large quantity. A smaller quantity of fibroblastlike cells from skin has also been established, partially characterized, and placed in frozen storage from the same fetus. This project is in support of the National Institute on Aging research in general cell biology. The present lines designated IMR-91 lung and IMR-91 skin complement the previous human diploid fibroblast culture (IMR-90) established from a female fetus. The lack of random inactivation of one of the two X chromosomes in the present male line reduces the genetic heterogeneity inherent in the female line.

The H1 histone is the least conserved of the five major groups of histone proteins. There are as many as five subtypes of H1 histone (1-3). These H1 subtypes occur in different amounts in different animal species and also show tissue specificity (1-3). Normal and neoplastic tissues from the same animal, e.g. rat or calf, contain the same H1 subtypes but in different relative amounts (4, 5). Because H1 subtypes exhibit tissue specificity, it is therefore difficult to identify the changes in their composition that are associated with neoplasia reported in studies utilizing tissues of different origins (4, 5). Tissue culture cells and their in vitro transformed neoplastic counterparts, on the other hand, offer an excellent system in which to study these changes because the cells are derived from the same origin. We have examined normal and neoplastic human cells and found a relationship between the H1 composition and the ability of the cells to form tumors in nude mice. The ratio of H1A to H1B in normal human cells is considerably lower than that in neoplastic cells and this ratio increases with the increased ability of the cells to form tumors in nude mice.

Human diploid cultures are widely used for the study of aging. We have developed quantitative cell culture procedures that include optimization of culture conditions and determination of replicative age by direct cell count. Using these procedures we are able to determine reproducibly the stage in the replicative life history of diploid cell cultures.

Exponentially growing and stationary phase young and old cultures of the human cell line WI-38 were studied using cytochemistry at the ultrastructural level. 5'-Nucleotidase activity was present in the plasma membrane of all cells examined; in exponentially growing cultures the reaction was more intense in mitotic cells than in interphase cells. An increase in the amount of the reaction product was observed at confluencey, especially in older cells. The reaction of Mg2+-activated adenosine 5'-triphosphatase was absent or very weak in exponentially growing cells and increased at confluency, especially in older cells. Alkaline phosphatase was detectable only in the cell membranes and in intercellular spaces of young cells at confluency. Acid phosphatase activity was increased in old cells, especially at confluency. In these old cells, positive reactions appeared in numerous small lysosomes, autophagic vacuoles and in some flattened sacs of the Golgi apparatus. The obtained results confirm and extend previous biochemical observations and indicate that changes in phosphatase activity are associated with proliferative activity and senescence of cells growing in vitro.