Treatment of the T98G glioblastoma cell line with antisense oligonucleotides directed toward mRNA encoding - Transforming growth factor-alpha and theepidermal growth factor receptor

Antisense oligonucleotides (oligos) complementary to mRNA encoding transfor ming growth factor-alpha (TGF-alpha) and its target, the epidermal growth f actor receptor (EGFR), are efficacious against human prostate and breast ca ncers carried in athymic nude mice. Glioblastomas, also regulated by EGFR e xpression, would appear to be similarly susceptible, and we now employ them against the T98G tumor model. T98G cells were distributed into wells and allowed to adhere prior to addit ion of oligos (12.5 muM) directed against TGF-alpha and/or EGFR for 6 d of treatment before thymidine radiolabeling. Supplemental media and oligos (25 muM final concentration) were added after d 3. Statistically significant i nhibition by oligos directed against TGF-alpha, EGFR, and their combination was 13.8%, 26.3%, and 18.1%, respectively. In a subsequent experiment cell s were incubated with increasing amounts of each oligo and their combinatio n for 3 d prior to radiolabeling. Statistically significant inhibition of g rowth for either oligo at every concentration was found. Cells incubated wi th 6.25, 12.5, 25, and 50 muM antisense directed against TGF-alpha had a me an inhibition of 29.3%, 33.3%, 21.7%, and 46.6%, respectively. Cells simila rly treated with oligos against EGFR had a mean inhibition of 77.9%, 80.3%, 82.0%, and 83.7%, respectively, and cells incubated with 6.25, 12.5, 25 an d 50 muM of each oligo had a mean inhibition of 74.7%, 70.6%, 70.8%, and 76 .3%, respectively. Lastly, in a paired experiment, cells treated with 0, 0. 39, 0.78, 1.56, 3.125, and 6.25 muM of oligos, either specifically directed against EGFR or a random control, for 3 d were evaluated for both thymidin e incorporation and EGFR expression. Statistically significant inhibition o f H-3-thymidine incorporation was seen in cells with the oligo specifically directed against EGFR at 3.125 muM and 6.25 muM when compared to non-oligo containing controls. This was accompanied by a comparable significantly de creased expression of a low-MW reactive derivative of EGFR at 3.125 muM and 6.25 muM in Western blots, and of a high-MW reactive EGFR at 6.25 muM. The significant effect against high-MW EGFR was observed vs both the non-oligo containing control and the random sequence. Oligo concentrations between 0 .78 and 1.5 muM also resulted in decreased expression of the low-MW form, b ut not significant differences in thymidine radiolabeling. In recovery expe riments, cells treated initially with greater oligo concentrations required significantly increased time to recover, particularly in cells treated wit h EGFR directed oligos. Intracellular uptake and nuclear localization was d emonstrated with FITC tagged oligos. In summary, even at relatively low oligo concentrations and short exposure, oligos against TGF-alpha, and particularly EGFR, significantly inhibit in vitro growth of the T98G glioblastoma, possibly mediated by decreased EGFR expression.