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Dissection of c-myc domains involved in S phase induction of NIH3T3 fibroblasts
S Goruppi1, S Gustincich, C Brancolini
1L.N.C.I.B., Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, Trieste, Italy.
Abstract:
The product of the c-myc proto-oncogene is an important regulator of cell proliferation and apoptosis in murine fibroblasts. Addition of the tumor promoter, phorbol myristate acetate (PMA), prevents apoptotic cell death induced by low serum concentrations in NIH3T3 cells that constitutively express and are transformed by v-myc. The protective effect of PMA allowed us to analyse the ability of normal c-Myc and Myc deletion mutants to induce serum starved, untransformed NIH3T3 cells to enter S phase. By microinjecting these quiescent cells with wild type and mutant human c-myc plasmids, we showed that full length c-myc is able to induce S phase entry in presence of PMA, but that c-Myc mutants that delete amino acids delta 7/91, delta 41/53, delta 56/103, delta 106/143, delta 265/317 and delta 414/433 are totally inactive. c-Myc did not shorten the period before entry into S phase, since Myc overexpressing cells entered S phase with the same kinetics as control cells when both were stimulated with 20% fetal calf serum (FCS). However, c-Myc overexpression did increase the percentage of cells entering S phase when these cells were stimulated with 2% fetal calf serum. Interestingly, this ability to enhance stimulation by a suboptimal concentration of FCS was retained to a significant degree by Myc mutants that delete amino acids delta 41/53, delta 56/103 or delta 265/317. Finally, Myc mutants that delete delta 106/143 or delta 414/433 exerted a dominant negative effect on S phase entry both in quiescent cells stimulated with 2% FCS and in unsynchronized, cycling cells.
Insights
The c-Myc protein regulates cell division. Specific mutations in c-Myc prevent it from driving cells into S phase, while others show a dominant negative effect on cell proliferation.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The c-Myc proto-oncogene product is a key regulator of cell proliferation and apoptosis.
- Tumor promoters like phorbol myristate acetate (PMA) can prevent apoptosis in certain cell lines.
- Understanding c-Myc's role in cell cycle progression is crucial for cancer research.
Purpose of the Study:
- To investigate the ability of wild-type c-Myc and its deletion mutants to induce quiescent NIH3T3 cells to enter S phase.
- To determine the functional domains of c-Myc required for cell cycle progression.
- To analyze the effect of c-Myc mutants on cell proliferation under suboptimal growth conditions.
Main Methods:
- Microinjection of wild-type and mutant human c-myc plasmids into serum-starved NIH3T3 cells.
- Use of PMA to prevent apoptosis and allow analysis of S phase entry.
- Stimulation of cells with varying concentrations of fetal calf serum (FCS).
Main Results:
- Full-length c-Myc induces S phase entry in the presence of PMA.
- Specific c-Myc deletion mutants (delta 7/91, delta 41/53, delta 56/103, delta 106/143, delta 265/317, delta 414/433) were found to be inactive in inducing S phase.
- c-Myc overexpression increased the percentage of cells entering S phase with suboptimal FCS (2%), an effect partially retained by mutants delta 41/53, delta 56/103, and delta 265/317.
- Mutants delta 106/143 and delta 414/433 exhibited a dominant negative effect on S phase entry.
Conclusions:
- The study identifies critical domains within c-Myc necessary for inducing cell cycle progression.
- Certain c-Myc mutants can interfere with the function of endogenous Myc proteins, demonstrating a dominant negative role.
- These findings contribute to understanding the complex regulation of cell proliferation by c-Myc and its mutants.