p14ARF induces G2 cell cycle arrest in p53- and p21-deficient cells by down-regulating p34cdc2 kinase activity
Authors: Dilek Güner
Jana Wendt
Berlinda Verdoodt
Guillaume Normand
Philipp G Hemmati
Bernd Dörken
Clarissa von Haefen
Peter T Daniel
Evelyne May
Journal: Journal of Biological Chemistry (2005)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: INK4a gene locus; tumor suppressor proteins; p14(ARF); p53-independent cell cycle regulation; G(2) arrest
Affiliations: Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Campus Berlin-Buch, D-13125 Berlin-Buch, Germany.

Abstract:  The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation. 

Retracted: No

PubPeer comments:
null, 
 Figure 6A. Much more similar and different than expected. ![file] , 
 Figure 2A. Much more similar than expected. ![file] , 
 #1 Figure 6A. Vertical, dark streaks or changes in background in p-cdc2 (Tyr-15) and cdc25C panels, but not in other panels. ![file] , 
 #1 The link above is not available anymore. Please see comment #4. #2 Publication of the two cited papers dates back to the year 2005. As can be easily seen by digital adjustments (contrast, brightness) the Western blots shown in the right panel (shows Figure 1b from Hemmati et al., Oncogene 2005) are uncut, i.e. in one piece. As stated on the front page of the paper, the manuscript was first submitted to Oncogene on June 14th 2004. At that point, the figure was already included and remained unchanged throughout the entire revision process up to final publication. The manuscript entitled u201cp14ARF induces G2 cell cycle arrest in p53- and p21-defienct cancer cells by down-regulating p34cdc2 kinase activityu201d (shown in the left panel) by G. Normand from our group was first submitted to The Journal of Biological Chemistry (JBC) at a later time point, i.e. on November 1st 2004. This is also shown on the front page of the published paper. Apparently, the Western blots shown there (Figure 6A) stem from the very same set of experiments (and their replicates). This may be seen easily by comparing the p53 bands. During assembly of Figure 6A, u00df-actin controls were obviously mixed up. The most plausible explanation is, that the blots were split u2013 a method commonly used at that time. Unfortunately, re-using some of the blots and mixing up controls was not uncovered by cross-checking the manuscripts before final submission or while editing the proofs. Partly re-using the Western blots in part in another publication was certainly not done on purpose but happened as a consequence of parallel preparation of the two papers by two separate authors. In any event, the publication of the work in Oncogene was first. JBC will be informed and the issue of how to handle partial double publication (concerning 2 other parameters) as well as splitting blots without proper mark-up will be addressed. #3 The histograms shown look identical. If so, a strongly assume that the Ad-p14ARF histogram was simply misplaced during copy paste retrieval of the pictures from the CellQuest analysis software of the flow-cytometer. I am quite confident, that this happened by accident, but certainly not on purpose. We will try to retrieve the correct histogram from the original data files. In any evet, thanks for posting the comment. #4 See comment above. P. G. Hemmati (still unable to create an account on pubpeer, sorry) ,