| Channel catfish leukocytes are highly unusual in that they readily undergo spontaneous in vitro immortalization yielding functionally active diploid cell lines that grow autonomously and express constitutively high levels of telomerase activity. In addition, nonautonomous, cell lines have been developed that require periodic restimulation for their continued proliferation. Initial studies were undertaken to determine when telomerase expression occurred during the immortalization process of catfish leukocytes. To this end, freshly isolated leukocytes from normal fish were shown to exhibit low to undetectable levels of telomerase activity. Within four days after culture initiation a dramatic increase in telomerase activity was observed which typically remained high for at least four weeks. This activity then declined, concomitant with decreases in proliferation and increases in cell death, Cells which escaped this “crisis” re-expressed high levels of telomerase activity indefinitely. Although telomerase activity was expressed early in the immortalization process, cells from clonal lines derived from these cultures had very short telomeres. Other studies demonstrated the expression of myc, nm23, hsp70, and p53 in catfish autonomous (immortal) and nonautonomous (mortal) leukocyte lines, as well as in normal stimulated leukocytes. These studies were facilitated by the previous identification of catfish hsp70 and p53 genes and the identification of catfish myc and nm23 genes reported here. It was found that channel catfish autonomous leukocyte lines, unlike normal resting PBL, constitutively express high levels of myc, nm23, hsp70, and truncated forms of p53 protein. On the other hand, catfish nonautonomous leukocyte lines transiently express myc, nm23, and hsp70 after stimulation with alloantigen. Stimulation with mitogens and exogenous cytokine factor(s) derived from catfish T cell supernatants, as well as alloantigen, also led to transient expression of telomerase activity in the nonautonomous cell lines. Both nonautonomous and autonomous cells display low and noncycling levels of p53 mRNA with multiple size forms of the p53 protein. These studies demonstrate that telomerase expression in catfish leukocytes is activation induced, and its expression does not stabilize telomere length until a critically short length is reached. In addition, it appears that truncation of p53 protein is not involved in the spontaneous in vitro immortalization process of catfish leukocytes. |
