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INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence
Sharon Brookes1, Janice Rowe, Margarida Ruas
1Molecular Oncology and Human Cytogenetics Laboratories, Cancer Research UK London Research Institute, Lincolns Inn Fields, London WC2A 3PX, UK.
Abstract:
The CDKN2A tumour suppressor locus encodes two distinct proteins, p16(INK4a) and p14(ARF), both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary fibroblasts from a member of a melanoma-prone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16(INK4a) deficient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14(ARF). Although they have a finite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We find that in human fibroblasts, ARF is not induced demonstrably by RAS, pointing to significant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.
Insights
Melanoma-prone cells lacking p16INK4a but retaining p14ARF resist RAS-induced senescence. Oncogenic RAS and hTERT enable anchorage-independent growth, revealing cell-type specific CDKN2A locus functions.
Area of Science:
- Oncology
- Cell Biology
- Genetics
Background:
- The CDKN2A tumor suppressor locus produces p16INK4a and p14ARF proteins, crucial for preventing uncontrolled cell proliferation.
- Replicative senescence is a permanent growth arrest mechanism in somatic cells, often triggered by proliferative stress or telomere shortening.
- Mutations in CDKN2A are linked to familial melanoma, highlighting its role in cancer predisposition.
Purpose of the Study:
- To investigate the functional consequences of a specific CDKN2A intragenic deletion in primary fibroblasts from a melanoma-prone individual.
- To determine the impact of p16INK4a deficiency and retained p14ARF function on cellular responses to oncogenic RAS.
- To explore the potential for cellular transformation and immortalization in these cells.
Main Methods:
- Analysis of primary fibroblasts with a homozygous intragenic CDKN2A deletion.
- Assessment of p16INK4a and p14ARF protein expression and function.
- Evaluation of cellular response to oncogenic RAS expression, including senescence induction.
- Investigation of anchorage-independent growth upon co-expression of RAS and telomerase (hTERT).
- Karyotyping and p53 functional analysis of resulting cell colonies.
Main Results:
- Fibroblasts were deficient in p16INK4a but expressed functional p14ARF.
- These cells exhibited resistance to RAS-induced growth arrest.
- Ectopic expression of RAS and hTERT led to the outgrowth of anchorage-independent colonies.
- These colonies maintained diploid karyotypes and functional p53.
- RAS did not demonstrably induce ARF in human fibroblasts, suggesting species- or cell-type specific regulation.
Conclusions:
- The CDKN2A locus imposes distinct proliferative barriers in different cell types or species.
- ARF's role in RAS-mediated senescence appears context-dependent.
- Understanding these mechanisms is crucial for comprehending tumor suppression and developing cancer therapies.