Tài liệu Báo cáo khoa học: Analyzing changes of chromatin-bound replication proteins occurring in

Analyzing changes of chromatin-bound replication proteins occurring

in response to and after release from a hypoxic block of replicon

initiation in T24 cells

Maria van Betteraey-Nikoleit, Karl-Heinz Eisele, Dirk Stabenow and Hans Probst

Physiologisch-Chemisches Institut der Universita¨t Tu¨bingen, Germany

It was shown previously [Riedinger, H. J., van Betteraey￾Nikoleit, M & Probst, H. (2002) Eur. J. Biochem. 269,

2383–2393] that initiation of in vivo SV40 DNA replication

is reversibly suppressed by hypoxia in a state where viral

minichromosomes exhibit a nearly complete set of repli￾cation proteins. Reoxygenation triggers fast completion

and post-translational modifications. Trying to reveal such

fast changes of chromatin-bound replication proteins in the

much more complex replication of the cellular genome

itself, we developed a protocol to extend these studies using

the human bladder carcinoma cell line T24, which was

presynchronized in G1 by starvation. Concomitantly with

stimulation of the cells by medium renewal, hypoxia was

established. This treatment induced T24 cells to contain a

large amount of replicons arrested in the ‘hypoxic preini￾tiation state’, ready to initiate replication as soon as normal

pO2 was restored. Replicons in other stages of replicative

activity were not detectable. Consequently the arrested

replicons were rapidly released into synchronous initiation

and succeeding elongation. Extraction of T24 nuclei with a

Triton X-100 buffer yielded a fraction containing the

cellular chromatin, including DNA-bound replication

proteins, while unbound proteins were removed. The use￾fulness of this protocol was tested by the proliferation

marker PCNA. We demonstrate here that this protein

switches from the remainder cellular protein pool into the

Triton-extracted nuclear fraction upon reoxygenation.

Employing this protocol, analyses of chromatin-bound

MCM2, MCM3, Cdc6 and cdk2 suggests that the ‘classi￾cal’ prereplication complex is already formed during

hypoxia.

Keywords: chromatin; DNA replication; hypoxia; nuclei;

synchronization.

Apart from control by cell cycle signals, DNA replication in

mammalian cells is subject to a regulation which depends on

the O2 tension in the cellular environment. Presumably, this

regulatory phenomenon, adapting the intensity of DNA

replication of growing cells to the supply of O2, is important

during embryonic growth and wound healing, and influen￾ces the propagation of malignant tumors. The O2-depend￾ent regulation concerns cells which are in S-phase or are

definitively committed to enter S-phase. When the concen￾tration of O2 drops to about 0.2–0.02%, scheduled replicon

initiations are suppressed and already-active replication

forks are slowed down. Of the cell lines examined so far,

only Ehrlich ascites cells exhibit suppression of replicon

initiation without a significant slowing down of fork

progression [1–3]. During hypoxia cells accumulate repli￾cons in a state ready to initiate (almost instantaneously)

within a few minutes after oxygen recovery. Thus, sudden

reoxygenation after several hours of hypoxia triggers a

synchronous burst of initiations of the accumulated repli￾cons followed by normal replication. So far, this regulatory

phenomenon has been published for Ehrlich ascites, HeLa

and CCRF cells [2,4,5]. Further cell lines examined so far,

e.g. T24, A549, PC3, TC7, BHK, SW2, HL60 and HUVEC

are also sub ject to the fast O2-dependent control of

replication (G. Probst, H. Probst & M. van Betteraey￾Nikoleit, unpublished results). We therefore suggest that it

represents a general phenomenon in mammalian cells,

although the molecular mechanisms involved are still largely

obscure. The remarkably fast resumption of initiations after

reoxygenation suggests that the O2-dependent replication

control acts very directly on the replication apparatus itself.

As published earlier, replication of the SV40 genome in

virus infected cells also obeys the oxygen-dependent regu￾lation [6,7]. Reoxygenation of virus infected cells after

several hours of hypoxia triggers a burst of hypoxically

accumulated viral replicon initiations followed by a syn￾chronous round of completely regular replication of viral

genomes.

Studying several replication proteins bound to SV40

minichromosomes before and after reoxygenation, i.e.

before and after triggering initiation, we found that a large

number of polypeptides taking part in viral replication were

bound to the SV40 minichromosome already under hypo￾xia. However, the multiprotein complexes necessary for

unwinding, primer synthesis and elongation lacked essential

components and remained incomplete as long as hypoxia

lasted [8]. Reoxygenation triggered fast completion to a

Correspondence to M. van Betteraey-Nikoleit, Physiologisch-Chem￾isches Institut der Universita¨t Tu¨bingen, Hoppe-Seyler-Straße 4,

D-72076 Tu¨bingen, Germany.

Fax: + 49 7071293339, Tel.: + 49 70712973329,

E-mail: [email protected]

Abbreviations: PCNA, proliferating cell nuclear antigen; BrdU,

5¢-bromodeoxyuridine; FITC, fluorescein isothiocyanate.

(Received 30 April 2003, revised 24 July 2003,

accepted 28 July 2003)

Eur. J. Biochem. 270, 3880–3890 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03769.x