Effects of an L-glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON), on the growth patterns of two human cervical carcinoma cell lines
Creators: name: Lang, Lye Mooi
Publisher: name: University of British Columbia
Year: 2010

description: The objective of this study was to observe the effects of a potential chemotherapeutic drug and an inhibitor of glycosaminoglycan synthesis, 6-diazo-5-oxo-L-norleucine (DON), on the growth patterns of two human cervical carcinoma cell lines. In particular, the in vitro cell-shedding patterns, colony formation and aggregation of the cell lines were examined with DON treatment, to test the hypothesis that secretion of extracellular materials (ECM) by one cell line (C-4II) was responsible for its more dispersed and infiltrative growth pattern. The cultured human cervical carcinoma cells used in this study were derived initially from the same biopsy specimen. These two morphologically different cell lines had distinctive patterns of growth both in vivo and in vitro. C-4I cells grew as round masses. In vivo, these cells did not infiltrate into host hamster tissues, while in vitro, few of these cells were shed from confluent cultures into culture medium. In contrast, C-4II cells did not grow as round cohesive masses, but in a dispersed manner. In vivo, these cells grew as small clumps and infiltrated into host tissues, while in vitro, such cultures shed many viable cells into culture medium. Previous ultrastructural analysis indicated that tumors with such dispersed growth patterns tend to have many features of secretory cells, with characteristics of glandular and basal cell differentiation. In this study, an L-glutamine analogue, DON, was used to inhibit the secretion of ECM. DON has been shown to inhibit the formation of many glutamine-requiring metabolites in the cell, including glucosamine-6-phosphate, a metabolite essential for the formation of ECM. Hence, the effects of DON on the growth and shedding patterns of the 2 types of human cervical cancer cells were examined. This study showed that DON had numerous morphological effects on cultured C-4 cells, other than its well-known growth-inhibitory effect. In addition, results showed that the shedding patterns of C-4 cells were altered with DON treatment. By harvesting cells shed into culture medium, it was determined that DON induced shedding in C-4I cultures while it enhanced shedding in confluent C-4II cultures. This increased shedding was most likely caused by a decrease in cell-cell cohesion. Furthermore, the aggregation of C-4I cells, treated with DON prior to dissociation, was greatly inhibited. Decreased cell-cell cohesion might also account for the decrease in stratification of C-4I cultures, irregularly-shaped C-4I colonies and aggregates, and for the "holes" that appeared within both types of C-4 colonies treated with high doses of DON. In contrast, aggregation of similarly treated C-4II cells was not consistently inhibited. Changes in colony forms, to more irregular shapes, were evident in both cell lines. By examining single cells growing on the substratum, it was determined that these changes might be due to DON decreasing cell-substratum adhesion in both cell lines. DON-treated C-4 cells were significantly larger when projected two-dimensionally. This was due, at least in part, to increases in cell volume, as detected on the Coulter counter and size distribution analyzer. The results of this study do not support the previous hypothesis that the more dispersed and infiltrative growth pattern of C-4II tumors, as compared to C-4I tumors, was due to the secretion of ECM. This hypothesis predicted a decrease in the shedding of viable cells and a change from a dispersed to a compact growth pattern in DON-treated cultures. Instead, the results indicate the contrary. The results suggest that cells may be secreting DON-sensitive cell surface-associated ECM that are responsible for the cohesive nature of C-4I cells. C-4II cells may produce less of this cohesive material, resulting in less cohesive cultures and more dispersed growth. Hence, with DON treatment, C-4I cultures were altered, resembling C-4II cultures, in terms of their shedding patterns, aggregabi1ity and morphology of aggregates and colonies.