Spontaneous fusion and formation of hybrids between C1300 neuroblastoma cells and lymphoid cells in mixed cultures

Abstract

Spleen lymphoid cells from specifically sensitized A/J mice were induced to bind and form rosettes around syngeneic, cloned C1300 neuroblastoma (NB) cells in vitro. Rosette formation usually led to target cell lysis. Electron microscopic evidence suggests, however, that lymphoid cells were sometimes spontaneously incorporated by the target cell and lysed, and their material was probably reutilized by the penetrated cell. When lymphoid cell suspensions were added to cultures of a mutant clone of NB cells (clone NA), which died in HAT medium because of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency, HAT medium-resistant clones arose at a frequency of about 5 × 10 −5, at least 200 times the reversion rate of NA. This suggested that correction of the HGPRT deficiency was due to efficient spontaneous fusion. Rosettes were also induced between spleen lymphocytes from allogeneic C 3H/HeJ mice and cloned NB cells. Rosettes were then separated by centrifugation through a discontinous BSA gradient. NB cells were isolated by adherency and were left to grow. Evidence indicated that these resulting NB cultures contained hybrids and that lymphocytes introduced new genes into NB cells with detectable frequency. Cells synthesized in culture a heteropolymer of glucose phosphate isomerase, while lymphocytes and original neuroblastoma cells alone supplied, respectively, only a fast and a slow form of the isozyme, as shown by electrophoretic assay. Furthermore, expression of hybrid surface markers, changes in lymphocyte recognition capacity in in vitro mixed cell culture assays, and delayed malignancy in A/J mice proved somatic cell hybrid formation.