Activation of Metabolic Systems During Tumor-Cell Formation.

Braunl-4 has clearly demonstrated that the transformation of normal plant cells into crown-gall tumor cells includes the progressive activation of enzyme systems which, once in operation, become a permanent part of the metabolic machinery of the altered cells. The work in our laboratory has paralleled that in Dr. Braun's with surprisingly similar results. We have, however, utilized a different technique, permitting us, in some particulars, to garner information not readily obtainable with his system and to determine some of the interrelations between the etiological agents acting on the cells and the activation of specific metabolic systems. A quantitative bioassay has been developed for these studies which utilizes the cambial-adjacent phloem tissues of carrot roots.' Tumor tissues formed in the surface of these disks can be weighed, giving a direct measure of the rates of duplication of tumor cells. We have delimited with some precision the temporal relations of the processes leading to tumor-cell formation in carrots, as well as the nature of the etiological agents (Fig. 1). Transformation of carrot phloem cells into tumor cells occurs in three phases, designated "conditioning," "induction," and "promotion." Optimal conditioning takes 16-20 hours and requires the action of several causal agents normally present in wound juice.2' 6, 7 The bacteria are unnecessary at this time. By pre-assay, carrot roots are obtained which are deficient in one of these agents,7 and these roots possess only limited tumor-forming capacity. Conditioning may also be limited by restricting the time of action of these agents, by interfering with their action with antimetabolites of the conditioning agents, or by slowing down the process with cold treatment. Induction takes 60-70 hours for completion and requires the action of a tumorinducing principle (T-iP) which may be a DNA synthesized by genetically competent crown-gall bacteria in the presence of a T-iP synthesis factor also present in the wound juice.8 Induction may be limited at will by using strains of crown-gall bacteria synthesizing a less effective T-iP or by holding the conditioned and inoculated disks for given periods of time during the induction phase at elevated temperatures9 where T-iP is presumably not synthesized by crown-gall bacteria. Incipient tumor cells formed by the action of conditioning and induction agents are promoted into primary tumor cells by auxins.10' 11 Promotion requires 28-30 hours in carrot. This phase has been inhibited by presenting an antiauxin to the tissues during the critical time. Tumors are macroscopically evident within 12 hours after the end of transformation, and their rates of duplication are linear for 10-15 days. For optimal transformation, the amounts, composition, and presentation times of all causal agents must be precisely regulated. Any deviation from the optimum results in the formation of slow-growing tumors. The exogenous stimulation of slow-growing tumors resulting from incomplete conditioning, induction, or promotion by specific growth factors may be taken as presumptive evidence that the