Development of resistance of novikoff hepatoma TO 5-fluoro-2'-deoxyuridine

Three strains of Novikoff hepatoma were studied in tissue culture; the N1-S1 or FUDR-sensitive strain, N1-S1/FUDR which is grown in and is resistant to 1 × 10−5M FUDR, and the cells which emerged as FUDR-resistant after 10 generations when N1-S1 was exposed to 1 × 10−7M FUDR. Inhibition of growth of N1-S1 cells by FUDR was related to the concentration of the drug in the medium. The uptake of formate and serine into DNA-thymine of these cells was inhibited by FUDR and the degree of inhibition was likewise proportional to the concentration of drug in the medium. The cells derived from N1-S1 which were resistant to FUDR resembled the stable resistant strain, N1-S1/FUDR, in that they lacked the ability to incorporate thymidine into DNA at 4.0 × 10−8M. Such cells apparently had less thymidine kinase activity and consequently also had a decreased ability to convert FUDR to the active monophosphate. Because of the decrease in thymidine kinase activity, the resistant strain, N1-S1/FUDR, utilized thymidine as a source for DNA-thymidine to a lesser extent than did the sensitive strain N1-S1. However, both strains used serine and formate as the source of the 5-methyl carbon of DNA-thymidine to a similar extent. Growth studies in shaker flasks with a mixture consisting of 75% N1-S1 and 25% N1-S1/FUDR cells treated with FUDR demonstrated that rapid overgrowth by the 25% resistant cells obscured the significant killing of sensitive cells by the drug. Clonal studies revealed that a small portion of cells resistant to FUDR were present in the sensitive N1-S1 strain of cells. The number of colonies which survived depended upon the concentration of the drug, but approximately 0.6% of the N1-S1 cells grew in the presence of 1 × 10−5M FUDR. These studies led to the conclusion that the development of drug resistance in this system was actually a process of selection by which FUDR killed a certain percentage of cells determined by its concentration. Biochemically resistant cells which were present as a small proportion of the original “sensitive” tumor continued to proliferate and ultimately emerged as the dominant population. DEVELOPPEMENT DE LA RESISTANCE DE L'HEPATOME DE NOVIKOFF A LA 5-FLUORO-2'-DESOXYURIDINE Trois souches d'hepatome de Novikoff ont ete etudiees en culture tissulaire: la souche N1-S1 sensible a la FUDR, la souche resiste Nl-Sl/FUDR, cultivee dans 1 X 10−5 M FUDR et les cellules qui sont devenues resistantes a la FUDR apres dix generations lorsque la souche Nl-Sl avait ete exposee a 1 X 10−7 M FUDR. L'inhibition du developpement des cellules Nl-Sl provoquee par la FUDR etait liee a la concentration du produit dans le milieu. L'incorporation de formiate et de serine a l'ADN-thymine de ces cellules etait inhibee par la FUDR et le degre d'inhibition etait egalement proportionnel a la concentration du produit dans le milieu. Les cellules derivees de la souche Nl-Sl qui etaient resistantes a la FUDR ressemblaient a la souche resistante stable, Nl-Sl/FUDR, en ce qu'elles ne pouvaient pas incorporer la thymidine a l'ADN a 4.0 X 10−8 M. L'activite de la thymidine kinase etait apparemment moindre dans ces cellules, qui n'avaient donc pas la měme aptitude a transformer la FUDR en monophosphate actif. En raison de la diminution de l'activite de la thymidine kinase, la souche resistante, Nl-Sl/FUDR, utilisait la thymidine comme source de l'ADN-thymine dans une mesure moindre que la souche sensible Nl-Sl. Toutefois, les deux souches utilisaient autant l'une que l'autre la serine et le formiate comme source du methyl-5 carbone de l'ADN-thymine. L'etude de la croissance cellulaire dans des shakers contenant un melange de 75% de cellules Nl-Sl et de 25% de cellules Nl-Sl/FUDR traitees a la FUDR a montre que la surcroissance rapide des 25% de cellules resistantes empěche de voir si les cellules sensibles sont detruites en grand nombre par le produit. Les etudes clonales ont revele qu'il y avait une faible quantite de cellules resistantes a la FUDR dans la souche sensible Nl-Sl. Le nombre de colonies survivantes depend de la concentration du produit, mais environ 0.6% des cellules Nl-Sl proliferent en presence de 1 × 10−5 M FUDR. Ces etudes ont mene a la conclusion que, dans ce systeme, le developpement de la resistance au produit est reellement un processus de selection par lequel la FUDR detruit un certain pourcentage de cellules determine par sa concentration. Les cellules resistantes du point de vue biochimique, qui representaient une faible partie de la tumeur “sensible” initiale, ont continue de proliferer et sont finalement devenues la population dominante.