Consistent patterns of changing hormone responsiveness during continuous culture of cloned rat calvaria cells

Abstract

The loss of responsiveness to hormones in cell populations is a fundamental problem in cell biology and aging. We have studied this process in cloned rat calvaria (RC) bone cell populations maintained in exponential growth in long-term culture in α-minimal essential medium supplemented with fetal bovine serum. At various times after cloning, the populations were tested for their ability to respond to parathyroid hormone (PTH), prostaglandin E₂ (PGE₂), and L-isoproterenol (IPT) with an increase in intracellular cAMP. Clone RCB 2.2, which was originally responsive to PTH but not PGE₂, maintained this characteristic throughout 14 mo of culture, after which PTH responsiveness was gradually lost and a concomitant increase in responsiveness to PGE₂ was observed. Subsequently, PGE₂ responsiveness was also lost; however, continued response to IPT indicated the presence of a hormone-sensitive adenylate cyclase. A similar pattern of hormone responsiveness was observed when a number of frozen stocks of RCB 2.2 cells were thawed and the cells were again maintained in continuous culture. That this pattern of phenotypic change was not unique to clone RCB 2.2 was verified by assessing the hormone responses in other independently selected clones. Although the precise time sequence for hormone response changes was not constant, in all cases the pattern of hormone response changes was similar: i.e., PTH response was always lost and PGE₂ response often first increased and then also was lost, despite the maintenance of response to IPT. These data indicate that clonal hormone-responsive populations can reproducibly give rise to unresponsive populations in an ordered series of phenotypic changes. Changes in response to the hormones tested did not appear to be concomitant with changes in other phenotypic properties, e.g., decrease in alkaline phosphatase activity.