Figure 4.
Figure 4. TF up-regulation cosegregates with increased malignancy of CRC cells. (A) Growth of HCT116 (○) and variant sublines HKh-2 (▪) and 379.2 (▴) in SCID mice. (B) Elevated TF mRNA expression was observed in HKh-2 tumors 50 days after injection of cells in vivo (top, middle); PCR-RFLP assay for detection of K-ras codon 13 mutation (bottom). The HKh-2 cell line used to establish tumors displayed a faint mutant K-ras band, suggesting the presence of rare revertant cells that sustained codon 13 mutations in the remaining undisrupted (wild-type) K-ras allele. Such cells were enriched in HKh-2 tumors, as indicated by the bright mutant K-ras band. TF immunohistochemical staining of (C) HKh-2 tumor, (D) HCT116 tumor, and (E) 379.2 tumor. Only rare, single TF+ cells (arrows in panel C) were observed in small early stage (day 7) HKh-2 tumors, whereas the greatest TF positivity was observed in 379.2 tumors, consistent with the levels of TF protein and mRNA expressed by these cells in vitro. (F) After sorting of cultured HKh-2 cells on the basis of TF positivity/negativity and injection into SCID mice, TF+ (TF-POS, ○) HKh-2 cells grew faster than the original HKh-2 (▪) cell population in vivo, whereas TF- (TF-NEG,▵) HKh-2 cells were relatively nontumorigenic. TF Northern blotting confirmed the purity of the sort (inset). Results from 1 of 2 independent sorting experiments are depicted. (G) The TF-POS cell subpopulation (▪) is enriched in revertant cells that express mutant K-ras, as indicated by the increased intensity of the mutant K-ras band (relative to the wild-type band, ▦). (H) Expression of dominant inhibitory Ras-N17 (▪) in mutant ras-expressing HCT116, HKh-TUM2, DLD-1, and A549 cells diminishes TF promoter activity relative to controls (▦). (I) Marked attenuation of TF promoter activity by pharmacologic inhibition of MEK1 (PD98059, 50 μM, ▪) downstream of Ras. This effect was observed in CRC cells harboring mutant K-ras (HCT116) and their derivatives with deleted p53 (379.2). ▦ indicates DMSO control. Error bars indicate standard deviation.

TF up-regulation cosegregates with increased malignancy of CRC cells. (A) Growth of HCT116 (○) and variant sublines HKh-2 (▪) and 379.2 (▴) in SCID mice. (B) Elevated TF mRNA expression was observed in HKh-2 tumors 50 days after injection of cells in vivo (top, middle); PCR-RFLP assay for detection of K-ras codon 13 mutation (bottom). The HKh-2 cell line used to establish tumors displayed a faint mutant K-ras band, suggesting the presence of rare revertant cells that sustained codon 13 mutations in the remaining undisrupted (wild-type) K-ras allele. Such cells were enriched in HKh-2 tumors, as indicated by the bright mutant K-ras band. TF immunohistochemical staining of (C) HKh-2 tumor, (D) HCT116 tumor, and (E) 379.2 tumor. Only rare, single TF+ cells (arrows in panel C) were observed in small early stage (day 7) HKh-2 tumors, whereas the greatest TF positivity was observed in 379.2 tumors, consistent with the levels of TF protein and mRNA expressed by these cells in vitro. (F) After sorting of cultured HKh-2 cells on the basis of TF positivity/negativity and injection into SCID mice, TF+ (TF-POS, ○) HKh-2 cells grew faster than the original HKh-2 (▪) cell population in vivo, whereas TF- (TF-NEG,▵) HKh-2 cells were relatively nontumorigenic. TF Northern blotting confirmed the purity of the sort (inset). Results from 1 of 2 independent sorting experiments are depicted. (G) The TF-POS cell subpopulation (▪) is enriched in revertant cells that express mutant K-ras, as indicated by the increased intensity of the mutant K-ras band (relative to the wild-type band, ▦). (H) Expression of dominant inhibitory Ras-N17 (▪) in mutant ras-expressing HCT116, HKh-TUM2, DLD-1, and A549 cells diminishes TF promoter activity relative to controls (▦). (I) Marked attenuation of TF promoter activity by pharmacologic inhibition of MEK1 (PD98059, 50 μM, ▪) downstream of Ras. This effect was observed in CRC cells harboring mutant K-ras (HCT116) and their derivatives with deleted p53 (379.2). ▦ indicates DMSO control. Error bars indicate standard deviation.

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