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Activation of MAP kinases in growth responsive pancreatic cancer cells
1Dept. Médecine, Faculté de Médecine, Université de Sherbrooke, Quebec, Canada.
Abstract:
The implication of MAP kinases in the proliferation control of pancreatic cancer cells is still unknown. This study was undertaken to examine the contribution of the p44/p42 and p38 MAP kinases in the mitogenic response to epidermal growth factor (EGF) and bombesin in human pancreatic cancer cells, MIA PaCa-2 and PANC-1. Data indicate that EGF and bombesin stimulated growth of both cell lines. In MIA PaCa-2 cells, EGF and bombesin stimulated the in gel activation of p38 while p44/p42 kinases exhibited high basal activity and no response to stimuli. Growth and p38 activation were inhibited by genistein, wortmannin, PD98059 and SB203580, specific inhibitors of tyrosine kinase, phosphatidylinositol 3-kinase, MEK-1 and p38 kinases, respectively. In PANC-1 cells, EGF and bombesin stimulated p42 in gel activation; p44 remained highly activated and unresponsive to stimuli and p38 did not respond. Stimulated growth and p42 activation were inhibited by genistein, wortmannin and PD98059. Estimation of MAPK activities with a specific anti-active MAP kinase antibody indicated, however, that EGF increased the intensity of the bands corresponding to p42 and p44 MAP kinases in both cell lines, indicating that the mitogenic factor can regulate MAP kinase activity. Data also pointed out that ATP is sufficient to increase MAP kinase activity within the in gel assay technique and may thus explain the discrepancies existing between the in gel assay data and those obtained with the anti-active MAP kinase antibody.
Insights
Mitogen-activated protein (MAP) kinases
Area of Science:
- Cell Biology
- Molecular Biology
- Oncology
Background:
- The role of MAP kinases in pancreatic cancer proliferation is not fully understood.
- Epidermal growth factor (EGF) and bombesin are mitogenic factors implicated in cancer growth.
Purpose of the Study:
- To investigate the involvement of p44/p42 and p38 MAP kinases in the response of pancreatic cancer cells to EGF and bombesin.
- To analyze the signaling pathways regulating cell proliferation in MIA PaCa-2 and PANC-1 cell lines.
Main Methods:
- Cell culture of human pancreatic cancer lines (MIA PaCa-2, PANC-1).
- Stimulation with EGF and bombesin.
- Analysis of MAP kinase activation (p44/p42, p38) using in-gel kinase assays and Western blotting with anti-active MAP kinase antibodies.
- Inhibition studies using specific kinase inhibitors (genistein, wortmannin, PD98059, SB203580).
Main Results:
- EGF and bombesin stimulated growth in both cell lines.
- In MIA PaCa-2 cells, p38 activation was stimulated, while p44/p42 showed high basal activity. Inhibitors blocked growth and p38 activation.
- In PANC-1 cells, p42 activation was stimulated, p44 was highly active, and p38 was unresponsive. Inhibitors blocked growth and p42 activation.
- Western blotting revealed EGF-induced activation of both p42 and p44 in both cell lines, contradicting in-gel assay findings for p44.
- ATP in the in-gel assay may artifactually increase MAP kinase activity, explaining discrepancies.
Conclusions:
- p38 MAP kinase is involved in MIA PaCa-2 cell proliferation stimulated by EGF and bombesin.
- p42 MAP kinase is involved in PANC-1 cell proliferation stimulated by EGF and bombesin.
- Discrepancies between in-gel assays and antibody-based detection highlight potential technical artifacts in MAP kinase activity assessment.