Previous studies have shown that decreased expression of the
reduced folate carrier (RFC) and increased expression of
dihydrofolate reductase (DHFR) are associated with intrinsic and acquired
methotrexate resistance, respectively, in
osteosarcoma (OS). It has also been shown in
colorectal cancer that E2F-1 expression correlates with
thymidylate synthase (TS) and, to a lesser extent, DHFR expression. To begin to investigate the regulation of DHFR and RFC expression in OS samples,
mRNA expression of E2F-1 and E2F-4 were measured in OS
tumor samples and related to DHFR, RFC, and TS
mRNA expression. Using fluorescent quantitative real-time PCR, 112 human OS patient samples were investigated for potential E2F-1/E2F-4:DHFR, E2F-1/E2F-4:RFC, and E2F-1/E2F-4:TS correlations. The expression ranges for each gene are as follows: DHFR, 0.02-33.13 (median = 0.20); RFC, 0.02-229.13 (median = 1.91); TS, 0.01-9.99 (median = 0.15); E2F-1, 0.05-69.07 (median = 0.52); and E2F-4, 0.24-52.35 (median = 1.45). Spearman correlation coefficients (r(s)) for E2F-1:DHFR, E2F-1:RFC, E2F-1:TS, E2F-4:DHFR, E2F-4:RFC, and E2F-4:TS were calculated to be 0.53, 0.63, 0.60, 0.41, 0.58, and 0.33, respectively (P < 0.001). On the basis of this data, moderate correlations exist between E2F-1/E2F-4 and DHFR, RFC, and TS. These results suggest E2F-1/E2F-4 may play a role in the regulation of RFC expression, which has not been reported previously. The
E2F transcription factors are also related to DHFR and TS expression in OS samples, suggesting a possible involvement in
methotrexate resistance. Although E2F
mRNA levels correlate with DHFR, RFC, and TS
mRNA expression, additional experiments are necessary to determine the direct effects of these
transcription factors and identify other
proteins that may influence this relationship.