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Induction by herbimycin A of contact inhibition in v-src-expressed cells
K Suzukake-Tsuchiya1, Y Moriya, M Hori
1Showa College of Pharmaceutical Sciences, Tokyo, Japan.
Abstract:
Herbimycin A, an inhibitor of pp60src tyrosin kinase, caused src oncogene-expressed cells to become sensitive to contact inhibition, but did not affect ras oncogene-expressed cells. The cell lines tested were temperature sensitive v-src- and temperature sensitive v-ras-integrated nontransformed rat kidney cell line (NRK) (srctsNRK and rastsNRK, respectively) and a wild-type v-src-integrated NIH3T3 (src3T3). srctsNRK cells in densely populated cultures (plated at 1.25 x 10(4) cells/cm2), grown at 33 degrees C in the presence of 0.45 micrograms/ml of herbimycin A, ceased the cell cycle at the G0-G1 stage within 2 days, and the cells showed normal morphology. Upon removal of herbimycin A, the quiescent cells resumed the cell cycle in concert with morphological alteration from 'normal' to 'transformed', and proceeded through the S and M stages successively in a synchronized manner. Cells in the late S stage, compared with those in other stages of the cell cycle, were more sensitive to the killing effect of 5-fluorodeoxyuridine. Such synchronism of the cell cycle was not observed with sparsely populated cultures (2.5 x 10(3) cells/cm2); the cells resumed their asynchronous growth after removal of herbimycin A, although their morphology returned to 'transformed' as in the experiment with the densely populated cultures. The induction by herbimycin A of contact inhibition in densely populated cultures was also observed with src3T3 (grown at 37 degrees C) but not with rastsNRK (grown at 33 degrees C).
Insights
Herbimycin A induces contact inhibition in src oncogene cells, halting their cell cycle. This effect was not observed in ras oncogene cells, highlighting a specific role for src in cell growth regulation.
Area of Science:
- Cell Biology
- Molecular Oncology
- Biochemistry
Background:
- The src oncogene is implicated in cellular transformation and loss of contact inhibition.
- Ras oncogene signaling pathways also contribute to uncontrolled cell proliferation.
- Herbimycin A is a known inhibitor of pp60src tyrosine kinase activity.
Purpose of the Study:
- To investigate the effect of Herbimycin A on contact inhibition in cells expressing src or ras oncogenes.
- To determine if Herbimycin A can restore normal cell cycle regulation in oncogene-transformed cells.
- To compare the sensitivity of different oncogene-expressing cell lines to Herbimycin A.
Main Methods:
- Treatment of temperature-sensitive v-src and v-ras integrated rat kidney cell lines (srctsNRK and rastsNRK) and wild-type v-src integrated NIH3T3 (src3T3) with Herbimycin A.
- Culturing cells at different densities (high and low) and temperatures (33°C and 37°C).
- Cell cycle analysis (G0-G1, S, M stages) and morphological assessment following Herbimycin A treatment and removal.
Main Results:
- Herbimycin A induced cell cycle arrest at the G0-G1 stage and normal morphology in densely populated srctsNRK and src3T3 cells.
- Removal of Herbimycin A led to synchronized cell cycle re-entry and morphological transformation in previously arrested cells.
- Ras oncogene-expressing cells (rastsNRK) did not exhibit contact inhibition in response to Herbimycin A.
- Cells in the late S stage were more sensitive to 5-fluorodeoxyuridine.
Conclusions:
- Herbimycin A specifically restores contact inhibition in src oncogene-expressing cells by arresting the cell cycle.
- The src oncogene, but not the ras oncogene, is a target for Herbimycin A-induced cell cycle regulation.
- Herbimycin A's ability to synchronize cell cycles offers potential for targeted therapies, particularly in late S-phase.