Biallelic mutations in p16(INK4a) confer resistance to Ras- and Ets-induced senescence in human diploid fibroblasts - PubMed (original) (raw)

. 2002 Dec;22(23):8135-43.

doi: 10.1128/MCB.22.23.8135-8143.2002.

Janice Rowe, Mark Harland, Sarah Drayton, Sharon Brookes, Chandra Gooptu, Patricia Purkis, Mike Fried, Veronique Bataille, Eiji Hara, Julia Newton-Bishop, Gordon Peters

Affiliations

Biallelic mutations in p16(INK4a) confer resistance to Ras- and Ets-induced senescence in human diploid fibroblasts

Thomas J Huot et al. Mol Cell Biol. 2002 Dec.

Abstract

The INK4a/ARF tumor suppressor locus is implicated in the senescence-like growth arrest provoked by oncogenic Ras in primary cells. INK4a and ARF are distinct proteins encoded by transcripts in which a shared exon is decoded in alternative reading frames. Here we analyze dermal fibroblasts (designated Q34) from an individual carrying independent missense mutations in each copy of the common exon. Both mutations alter the amino acid sequence of INK4a and functionally impair the protein, although they do so to different degrees. Only one of the mutations affects the sequence of ARF, causing an apparently innocuous change near its carboxy terminus. Unlike normal human fibroblasts, Q34 cells are not permanently arrested by Ras or its downstream effectors Ets1 and Ets2. Moreover, ectopic Ras enables the cells to grow as anchorage-independent colonies, and in relatively young Q34 cells anchorage independence can be achieved without addition of telomerase or perturbation of the p53 pathway. Whereas ARF plays the principal role in Ras-induced arrest of mouse fibroblasts, our data imply that INK4a assumes this role in human fibroblasts.

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Figures

FIG. 1.

FIG. 1.

Family pedigree and nature of CDKN2A mutations. (A) The proband is indicated with an arrow, and the shaded symbols refer to individuals diagnosed with cancer (Ca) as follows: melanoma, filled symbol; lung cancer, left half shaded; rectal cancer, right half shaded; breast cancer, top half shaded. To preserve the confidentiality of the patient and family, genders and unaffected siblings are not specified in the pedigree. (B) Schematic representation of the CDKN2A locus with the exons shown as boxes. The sequences encoding p16INK4a are identified by stippling, and those encoding p14ARF are identified by cross-hatches. The nucleotide sequence of the relevant sections of exon 2 and the consequences for translation in the INK4a and ARF reading frames are shown at the bottom.

FIG. 2.

FIG. 2.

Functional analyses of p16INK4a variants in Q34 cells. (A) Lysates from Q34 cells and the control fibroblasts, 904, previously infected with a retrovirus encoding simian virus 40 T antigen, were immunoprecipitated with polyclonal antibodies against Cdk4 or p16INK4a as indicated. Following SDS-12% PAGE the amounts of Cdk4 and p16INK4a in each precipitate were compared by immunoblotting with the corresponding monoclonal antibodies. IP, immunoprecipitation. (B) Longer exposures are shown of Cdk4 and Cdk6 immunoprecipitates that have been immunoblotted for p16INK4a and the respective Cdks. (C) Normal HDFs were infected with recombinant retroviruses encoding 2× HA-tagged p16INK4a (wild type [WT]), and the indicated variants and pools of infected cells were selected in puromycin. Seven days after infection, cell lysates were prepared and equivalent amounts (500 μg) of protein were immunoprecipitated with antibodies against Cdk4 or Cdk6 (upper two panels). Immunoprecipitated proteins were fractionated by SDS-12% PAGE and were immunoblotted with a monoclonal antibody against p16INK4a. The positions of exogenous (2×HA p16) and endogenous p16INK4a (End p16) are indicated. Samples (20 μg) of total lysate from the same cells were analyzed directly by SDS-PAGE and immunoblotting for p16INK4a and Cdk4 (lower panels). (D) Aliquots of each cell pool, at 7 days postselection, were transferred into 24-well plates (5 × 103 cells/well), and their proliferation was monitored for 10 days. At each time point cells were fixed in 10% formaldehyde and viable cells were stained with crystal violet. Relative numbers of cells were determined by measurements of the optical density at 590 nm. The data present the averages of triplicate measurements.

FIG. 3.

FIG. 3.

Functional analysis of p14ARF in Q34 cells. (A and B) U20S cells were transfected with plasmids encoding MDM2, p53 and p14ARF as indicated. (A) Samples (500 μg of protein) of cell lysate were immunoprecipitated with a polyclonal antibody against p14ARF (JR14) and were immunoblotted with antibodies against p14ARF (4C6/4), MDM2 (IF2), and p53 (DO-1). (B) The p53 and MDM2 levels were analyzed by direct immunoblotting of cell lysate. (C) Control (Hs68) and Q34 HDFs expressing E2F1-ER were treated with (+) or without (−) 4-hydroxy tamoxifen (OHT) for 24 h. Samples (20 μg) of cell lysate were fractionated by SDS-PAGE and were immunoblotted with an antiserum raised against amino acids 54 to 75 of human p14ARF and with monoclonal antibodies against MDM2 and p53. Cdk4 served as a loading control. Samples (1 mg) of each lysate were immunoprecipitated with the same p14ARF antiserum and were immunoblotted for MDM2. IP, immunoprecipitation; WT, wild type.

FIG. 4.

FIG. 4.

Induction of p16INK4a by Ras, Ets1, and Ets2. Q34 HDFs and the Hs68 controls infected with retroviruses encoding Ras, Ets1, or Ets2 as indicated or with the empty vector (Vec) were selected in puromycin for 5 days. Samples (20 μg) of cell lysate were analyzed by SDS-12% PAGE and were immunoblotted with antisera against p16INK4a, Ras, Ets1, Ets2, and MEK as indicated.

FIG. 5.

FIG. 5.

Contrasting effects of Ras, Ets1, and Ets2 in Hs68 and Q34 cells. Pools of Hs68 and Q34 cells infected with retroviruses encoding Ras, Ets1, or Ets2 were photographed at 10 days postinfection. Whereas the Hs68 cells had undergone a senescence-like growth arrest (upper panels), the Q34 cells continued to proliferate, with only rare cells adopting an enlarged phenotype (lower panels).

FIG. 6.

FIG. 6.

Characterization of anchorage-independent Q34 cells. (A) Selected anchorage-independent colonies of Q34 cells expressing Ras alone (QR2 and QR3) or hTERT and Ras (QTR1 to QTR6) were analyzed for expression of key proteins. For comparison, similar analyses were conducted on pools of Q34 cells expressing hTERT (QT) and either Ras or empty vector. Samples (20 μg) of lysate were fractionated by SDS-PAGE in 8 and 12% gels and were immunoblotted for hTERT, Ras, p53, and Cdk4. (B) The indicated cell pools and agar colonies were exposed to UV irradiation (15 J/m2), and samples (20 μg) of total protein were subjected to SDS-PAGE and were immunoblotted for p53, p21CIP1, and Cdk4.

References

    1. Ashcroft, M., and K. H. Vousden. 1999. Regulation of p53 stability. Oncogene 18**:**7637-7643. -PubMed
    1. Bates, S., A. C. Phillips, P. A. Clark, F. Stott, G. Peters, R. L. Ludwig, and K. H. Vousden. 1998. p14ARF links the tumour suppressors RB and p53. Nature 395**:**124-125. -PubMed
    1. Brookes, S., J. Rowe, M. Ruas, S. Llanos, P. A. Clark, M. Lomax, M. C. James, R. Vatcheva, S. Bates, K. H. Vousden, D. Parry, N. Gruis, N. Smit, W. Bergman, and G. Peters. 2002. INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence. EMBO J. 21**:**2936-2945. -PMC -PubMed
    1. Brotherton, D. H., V. Dhanaraj, S. Wick, B. L., P. J. Domaille, E. Volyanik, X. Xu, E. Parisini, B. O. Smith, S. J. Archer, M. Serrano, S. L. Brenner, T. L. Blundell, and E. D. Laue. 1998. Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d. Nature 395**:**244-250. -PubMed
    1. Drayton, S., and G. Peters. 2002. Immortalisation and transformation revisited. Curr. Opin. Genet. Dev. 12**:**98-104. -PubMed

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