Permanent cell lines from erythrophoromas in goldfish (Carassius auratus)

Attempts to establish permanent cell lines from spontaneous erythrophoromas (tumors derived from red pigment cells or erythrophores) of goldfish were made with the use of biopsy specimens from 12 tumors in 10 fish. Three cell lines were established that grew in vitro in synthetic medium (L-15 or Dulbecco's modified Eagle's minimum essential medium) supplemented with 20% fetal bovine serum for more than 8 months. One of these lines (GEM-81) with high proliferative activity was cultured for over 200 generations without an obvious change in growth rate. From this cell line, clonal cultures were obtained that formed clones with relatively intense yellow pigmentation. Descendants of these cell lines and clones contained low but detectable amounts of pteridine pigments (such as 7-hydroxybiopterin, biopterin, xanthopterin, and isoxanthopterin) and numerous cytoplasmic organelles analogous to pterinosomes. Both these characteristics are phenotypic markers of normal erythrophores and their neoplastic counterparts. After numerous subcultivations, these long-term cultures differed from those of the initial explants in having much lower contents of total pteridines and relatively lowered contents of 7-hydroxybiopterin. As manifestations of their neoplastic origins, all cell lines examined showed disoriented growth with dense focal mounding in monolayer cultures. The population doubling time of the uncloned GEM-81 cell line was 31 hours at 35 degrees C over a feeder cell layer and 3 days at 25 degrees C without feeder cells. Injection of cultured cells (1.5 x 10(7) cells/fish) into normal goldfish that had not been immunosuppressed did not give rise to tumors within 5 months.