Tài liệu Báo cáo khoa học: Enhanced expression of Mcm proteins in cancer cells derived from uterine

Enhanced expression of Mcm proteins in cancer cells derived

from uterine cervix

Yukio Ishimi1

, Isao Okayasu2

, Chieko Kato1

, Hyun-Ju Kwon1

, Hiroshi Kimura3

, Kouichi Yamada4

and Si-Young Song1

1

Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo; 2

Department of Pathology, Kitasato University School of Medicine,

Sagamihara, Kanagawa; 3

Department of Functional Genomics, Medical Research Institute, Tokyo Medical and Dental University,

Tokyo; 4

National Institute of Health and Nutrition, Tokyo Japan

Minichromosome maintenance proteins (Mcm) 2–7 play

essential roles in eukaryotic DNA replication. Several

reports have indicated the usefulness of Mcm proteins as

markers of cancer cells in histopathological diagnosis.

However, their mode of expression and pathophysiological

significance in cancer cells remain to be clarified. We com￾paredthe level of expression ofMcm proteins among human

HeLa uterine cervical carcinoma cells, SV40-transformed

human fibroblast GM00637 cells andnormal human fibro￾blast WI-38 cells. All the proteins examinedwere detectedin

HeLa andGM cells at 6–10 times the level foundin WI-38

cells on average. This increase was observedboth in total

cellular proteins andin the chromatin-boundfraction.

Consistently, Mcm2 mRNA was enrichedin HeLa cells to

approximately four times the level in WI-38 cells, andthe

synthesis ofMcm4, 6 and7 proteins was acceleratedin HeLa

cells. Immunohistochemical studies of surgical materials

from human uterine cervix showedthat Mcm3 and4 are

ubiquitously expressedin cancer cells. Further, the positive

rate andlevel of Mcm3 and4 expression appearedto be

higher in cancer cells than in normal proliferating cells of the

uterine cervix anddysplastic cells, suggesting that they can be

useful markers to distinguish these cells.

Keywords: cancer cells; DNA replication; Mcm; protein

expression; uterine cervix.

The entire Mcm family (Mcm2–7) is essential for eukaryotic

DNA replication [1–4], playing roles in the initiation and

elongation of DNA replication [5]. Mcm2–7 proteins

constitute the prereplicative complex that is formedat the

replication origin [6,7]. Among several Mcm complexes,

only the Mcm2–7 hexamer has the ability to induce DNA

replication in Xenopus egg extracts [8]. All family members

have a DNA-dependent ATPase motif in the central

domain [9]. However, it has been reported that Mcm4, 6,

and7 form a hexameric complex andfunction as a DNA

helicase in vitro [10–13], suggesting that the Mcm4/6/7

complex acts as a DNA-unwinding enzyme in the replica￾tion. The exact biochemical function of Mcm2, 3, and5

remains to be determined, but it has been shown that these

proteins can inhibit the helicase activity of Mcm4/6/7 by

disassembling this hexamer [12,14,15], indicating a regula￾tory role. In vivo findings suggest that Mcm2–7 proteins act

as a replicative helicase that is responsible for fork

movement [5,7]. Thus, it is likely that the Mcm2–7 complex

is involvedin DNA replication as a DNA helicase, andan

activatedform of the Mcm2–7 complex is a Mcm4/6/7

hexamer.

Mcm proteins were identified as a component of the

DNA replication licensing system by which a single round

of DNA replication in a cell cycle is ensured[16–18]. It has

been shown that cyclin-dependent kinase plays a central role

in preventing over-replication [19]. Cdc6, involved in

loading Mcm proteins onto chromatin, is one of the targets

of regulation by the kinase [3]. Recently it has been shown

that deregulation of Cdc6, ORC (origin recognition com￾plex) andMcm, all of which are targets of phosphorylation

by cyclin-dependent kinase, leads to over-replication in

Saccharomyces cerevisiae [20]. These findings indicate that

Mcm proteins play a role in regulating the replication of

DNA. The gene amplification that has been detected in

various cancer cells [21] is probably generatedby the over￾replication of a genomic locus containing replication origins

[22,23]. These notions suggest that the deregulation of DNA

replication contributes to the development of malignant

transformation of cells.

Recently, several groups have reportedthat Mcm

proteins are more frequently detected in cells from malig￾nant tissues than those from normal tissues [24–33]. This

phenomenon was also observedin dysplastic cells, suggest￾ing that Mcm proteins are a goodindicator of proliferative

or cancer cells in malignant tissues [28]. Elucidation of how

the expression of Mcm protein changes relates to malignant

transformation of cells andthe pathophysiological signifi￾cance of those changes awaits further studies. In this paper,

we report that Mcm proteins are expressedat higher levels

in the transformedcell lines than in normal fibroblasts. We

also foundmore frequent andhigher-level expression of

Correspondence to Y. Ishimi, Mitsubishi Kagaku Institute of Life

Sciences, 11 Minamiooya, Machida, Tokyo 194–8511, Japan.

Fax: + 81 42 724 6314, Tel.: + 81 42 724 6266,

E-mail: [email protected]

Abbreviations: BrdU, bromodeoxyuridine; CIS, carcinoma in situ;

Mcm, minichromosome maintenance protein; ORC, origin

recognition complex.

(Received24 September 2002, revised16 December 2002,

accepted20 December 2002)

Eur. J. Biochem. 270, 1089–1101 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03440.x