Figure 1.
Mutant K-ras and p16shRNA were stably expressed in HPNE cells.
(A) The strategy for introducing gene alterations into HPNE cell lines. (B) Stable expression of mutant K-RasG12V in the HPNE cell line, as identified by Ras activity assay and Western blot analysis of total Ras expression. (C) The expression levels of K-ras and p16 in HPNE/K-ras cells were identified by RT-PCR analysis. (D) Stable expression of mutant K-ras and p16shRNA in HPNE cell lines. The stable cell lines were identified by Western blot analysis of the expression of RasG12V, total Ras and p16. (E) Western blot analysis of p-Rb and Rb expression in HPNE cell lines. (F) Western blot analysis of p14 expression in HPNE cell lines.
Figure 2.
Activation of K-ras and silencing of p16 in HPNE cells increased cell proliferation and growth.
(A) The cell growth curve of HPNE cell lines as detected by using a cell counter. (B) Cell cycle analysis of HPNE cell lines. The percentage of cells in each phase of the cell cycle is shown. (C) Anchorage-independent cell growth of HPNE cell lines in soft agar assay. A representative field of soft agar for each cell line is shown. (D) Cells were incubated with gemcitabine for 72 h, and cell viability was measured by MTT assay. (E) The expression of cell cycle proteins cyclins and c-Myc was increased by mutant K-ras in HPNE cell lines, as determined by Western blot analysis. β-Actin was used as loading control. (F) Relative mRNA levels of E2F target genes in HPNE/K-ras and HPNE/K-ras/p16sh cells. All data are presented as mean ± SD (n = 3 independent experiments). **P<0.01 versus the corresponding control groups.
Figure 3.
Activation of K-ras in HPNE cells increased cell invasion.
(A) The quantification of stained HPNE cell lines per field in a transwell-matrigel penetration assay. Representative micrographs for each cell line are shown. Data are presented as mean ± SD (n = 3 independent experiments). **P<0.01 versus the HPNE control cells. (B) The expression of EMT markers cytokeratin-19, E-cadherin, vimentin, and N-cadherin was detected in HPNE cell lines by Western blot analysis. (C) The expression of invasion-related proteins MMP2 and uPA in HPNE cell lines was detected by Western blot analysis. β-Actin was used as loading control.
Figure 4.
Knocking down of p16 enabled the HPNE/K-ras cells to overcome senescence.
(A) Representative fields of senescence-associated β-galactosidase (SA-β-gal) activity in HPNE cell lines. (B) Quantification of SA-β-gal staining in HPNE cells, data are showed as percentage of positive cells **P<0.01 versus the corresponding control groups. (C) The expression of cyclin-dependent inhibitors – p15, p21 and p27 was analyzed by Western blot in HPNE cell lines. β-Actin was used as loading control.
Figure 5.
HPNE cells with activation of K-ras and inactivation of p16 induced tumorigenesis in vivo.
(A) In vivo bioluminescence imaging of tumor growth by HPNE, HPNE/K-ras and HPNE/K-ras/p16shRNA cells at 8 weeks' after injection in NOD/SCID mice is shown. (B) The rates of tumor formation and metastasis in NOD/SCID mice. (C) Representative micrographs showing the histology of the orthotopic tumors formed by HPNE/K-ras/p16shRNA cells as revealed by H&E staining: (i) undifferentiated ductal carcinoma with sarcomatoid features, (ii) necrosis, (iii) liver metastasis, and (iv) spleen metastasis. (D) Immunohistochemical analysis of the expression of HER-2 and EGFR in tumors formed in vivo by the HPNE/K-ras/p16shRNA cells compared with those in human pancreatic cancer and human normal pancreatic duct. Scale bar: 100 µm.
Figure 6.
Molecular analysis of HPNE cell lines offers insight into malignant transformation in human pancreatic cancer.
(A) Activation of the Akt signaling pathway by K-ras in HPNE cell lines as detected by Western blot analysis. (B) Activation of the MAPK signaling pathway by K-ras in HPNE cell lines. The expression of total and phosphorylated-Erk and p38 was detected by Western blot analysis. β-Actin was used as loading control. (C) Expression of p16 was decreased after siRNA depletion of HBP1, as detected by real-time PCR. (D) Relative quantification of SA-β-gal staining cells after siRNA depletion of HBP1 in HPNE/K-ras cells (E) Expression of EGF and TGFα was increased in HPNE/Kras cells as detected by real-time PCR. (F) Expression of TGFα was increased in HPNE-iKras cells as detected by real-time PCR. (G) The proposed model for transformation of HPNE cell line. hTERT enabled cell to acquire replicating immortality; oncogenic K-ras rendered HPNE cells able to sustain proliferative signaling, and thus increased cell proliferation and cell growth; and inactivation of p16 by shRNA silencing enabled HPNE cells to evade growth suppressors, disrupt the senescence checkpoint, and ultimately induce transformation of HPNE cells.