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Non-malignant and tumor-derived cells differ in their requirement for p27Kip1 in transforming growth
Jeffrey C H Donovan1, Jeffrey M Rothenstein, Joyce M Slingerland
1Department of Molecular and Cell Biology, Sunnybrook & Women's College Health Sciences Centre, Toronto, Ontario M4N 3M5, Canada.
Abstract:
Transforming growth factor beta (TGF-beta) induces G(1) arrest in susceptible cells by multiple mechanisms that inhibit the G(1) cyclin-dependent kinases (Cdks), including Cdk2, Cdk4, and Cdk6. TGF-beta treatment of early passage finite lifespan human mammary epithelial cells (HMECs) led to an accumulation of p27(Kip1) in cyclin E1-Cdk2 complexes and kinase inhibition. The requirement for p27 in the G(1) arrest by TGF-beta was assessed by transfection of antisense p27 (ASp27) oligonucleotides into TGF-beta-treated HMECs. Despite a reduction in total and cyclin E-Cdk2 bound p27 after ASp27 transfection, HMECs remained arrested in the G(1) phase. Maintenance of the G(1) arrest was accompanied by increased association of the Cdk inhibitor p21(WAF-1/Cip-1) and the retinoblastoma family member p130(Rb2) in cyclin E1-Cdk2 complexes along with kinase inhibition. In contrast to the findings in HMECs, p27 was essential for G(1) arrest by TGF-beta in two tumor-derived lines. ASp27 transfection into two TGF-beta-responsive, cancer-derived lines was not associated with increased compensatory binding of p21 and p130 to cyclin E1-Cdk2, and these cell lines failed to maintain G(1) arrest despite the continued presence of TGF-beta. Progressive cell cycle deregulation leading to impaired checkpoint controls during malignant tumor progression may alter the role of p27 from a redundant to an essential inhibitor of G(1)-to-S phase progression.
Insights
Transforming growth factor beta (TGF-beta) arrests cells in G(1) phase. While p27 is not essential in normal mammary cells due to compensatory inhibitors, it is crucial for TGF-beta-induced G(1) arrest in cancer cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Transforming growth factor beta (TGF-beta) is a key regulator of cell growth and differentiation.
- TGF-beta induces cell cycle arrest at the G(1) phase through inhibition of cyclin-dependent kinases (Cdks).
- The role of specific Cdk inhibitors, such as p27(Kip1), in mediating TGF-beta-induced arrest can vary between cell types.
Purpose of the Study:
- To investigate the role of p27(Kip1) in TGF-beta-induced G(1) cell cycle arrest in human mammary epithelial cells (HMECs) and cancer-derived cell lines.
- To determine if other Cdk inhibitors compensate for the loss of p27 function.
- To understand how cell cycle deregulation in cancer affects the requirement for p27 in TGF-beta signaling.
Main Methods:
- Treatment of HMECs and cancer cell lines with TGF-beta.
- Transfection with antisense p27 (ASp27) oligonucleotides to reduce p27 levels.
- Analysis of cell cycle progression using G(1) arrest.
- Assessment of Cdk inhibitor complex formation (p27, p21, p130) with cyclin E1-Cdk2.
- Measurement of Cdk kinase activity.
Main Results:
- In HMECs, TGF-beta induced G(1) arrest and p27 accumulation in cyclin E1-Cdk2 complexes. However, reducing p27 levels did not prevent G(1) arrest, as p21(WAF-1/Cip-1) and p130(Rb2) compensated by inhibiting cyclin E1-Cdk2.
- In contrast, p27 was essential for TGF-beta-induced G(1) arrest in two cancer-derived cell lines.
- ASp27 transfection in cancer cells did not lead to compensatory binding of p21 or p130, and these cells failed to maintain G(1) arrest.
Conclusions:
- The requirement for p27 in TGF-beta-mediated G(1) arrest is context-dependent, being redundant in normal HMECs but essential in certain cancer cells.
- Cell cycle deregulation during malignant progression may shift the role of p27 from a redundant to a critical inhibitor of G(1)-to-S phase progression.
- These findings highlight the complex interplay of cell cycle regulators and their altered functions in cancer.