TAILIEUCHUNG - Báo cáo khoa học: Cdk2 activity is dispensable for triggering replicon initiation after transient hypoxia in T24 cells

We examined whether the fast release of replicon initiation after sudden O2 recovery of hypoxically incubated mammalian cells depends on kinase activity of Cdk2. We used a system based on starved⁄refed T24 cells elab-orated previously for such investigations [van Betteraey-Nikoleit M, Eisele KH, Stabenow D & Probst H (2003)Eur J Biochem270, 3880–3890]. | ềFEBS Journal Cdk2 activity is dispensable for triggering replicon initiation after transient hypoxia in T24 cells Dirk Stabenow Hans Probst and Maria van Betteraey-Nikoleit Interfakultares Institut fur Biochemie der Universitat Tubingen Germany Keywords hypoxia reoxygenation Cdk2 replication chromatin Correspondence M. van Betteraey-Nikoleit Physiologisch-Chemisches Institut der Universitat Tubingen Hoppe-Seyler-StraBe 4 D-72076 Tubingen Germany Fax 49 7071293339 Tel 49 70712973329 E-mail Received 6 July 2005 revised 16 August 2005 accepted 5 September 2005 doi We examined whether the fast release of replicon initiation after sudden O2 recovery of hypoxically incubated mammalian cells depends on kinase activity of Cdk2. We used a system based on starved refed T24 cells elaborated previously for such investigations van Betteraey-Nikoleit M Eisele KH Stabenow D Probst H 2003 Eur J Biochem 270 3880-3890 . Cells subjected to hypoxia concurrently with refeeding accumulate the G1 DNA content within 5-6 h. In this state they are ready to perform within 12 min after O2 recovery a burst of replicon initiations that marks the start of a synchronous S-phase. We found that Cdk2 binds to the chromatin fraction within 4-6 h after refeeding with fresh medium irrespective of whether the cells were incubated normoxically or hypoxically. However inhibition of Cdk2 by olomoucine roscovitine or the Cdk2 cyclin inhibitory peptide II had no influence on the synchronous burst of replicon initiations. Cdc6 and pRb possible targets of Cdk2 phosphorylation behaved differentially. Inhibition did not affect phosphorylation of Cdc6 after reoxygenation whilst chromatin bound pRb remained hypophosphorylated beyond the initiation burst. Thus neither Cdk2 activity though present at the end of the hypoxic period nor pRb phosphorylation are necessary for releasing the burst of replicon initiations upon oxygen recovery. .

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