British zyxwvutsrqpon Journal zyxwvutsrqponm of Haematoloau. 1995. 90, 94-99 zyxwvutsrq Over-expression and amplification of the CDC2 gene in leukaemia cells YUSUKE FURUKAWA, YASUHITO TERUI, KUMI SAKOE, MASATSUGU OHTA, SEIICHI KITAGAWA, YASUSADA MIURA AND MASAKI SAITO Division of Haemopoiesis, Institute of Haematology and Department of Haematology, Jichi Medical School, Tochigi, Japan Received 31 August 1 9 9 4 ; accepted for publication 2 5 January 1 9 9 5 Summary. The expression and structure of the cdc2 gene, one of the master regulators of the eukaryotic cell cycle, were investigated in fresh leukaemic cells from 51 cases of various types of leukaemia. Cdc2 mRNA transcripts were detectable in approximately zyxwvutsr 40% (21/5 1) of cases by Northern blotting. Over-expression of cdc2 mRNA as compared to normal bone marrow cells was noted in 10/21 cases with detectable cdc2 mRNA transcripts. Amplification of the cdc2 gene was found in three cases. Cdc2 mRNA was over-expressed in these three cases, suggesting that gene amplification is a direct cause of mRNA over-expression in a subset of cases. Cell proliferative capacity was well correlated with the amount of cdc2 mRNA transcripts, i.e. 'H-thymidine incorporation was highest in cases with cdc2 mRNA over-expression and was significantly higher in cdc2-positive cases than in cdc2- negative cases. These results suggest that over-expression of CDC2, which is due to the gene amplification in some cases, might play a role in altered growth of leukaemic cells. Keywords: CDC2, leukaemia, gene expression, gene amplification,cell cycle. The cdc2 gene was originally identified in fission yeast as an essential cell cycle control element for both Gl/S and G2/M transition (Nurse zyxwvutsrq & Bissett, 1981). It encodes a serinel threonine kinase designated as ~34'~'' which is now known to be a catalytic subunit of the mitosis-regulating protein kinase complex known as maturation promoting factor (MPF) (Dunphy et al, 1988; Gautier et zyxwvutsrq al, 1988). Both biochemical and genetic experiments implicate an involve- ment of CDC2 homologue in the regulation of mitotic initiation in mammalian cells (Draetta & Beach, 1988; Riabowol et al, 1989), whereas other members of cyclin- dependent kinases (CDKs),especially CDK2 and CDK4, have now been revealed to mediate G1 to zyxwvut S transition (Quelle et al, 1993; Ohtsubo & Roberts, 1993). Post-translational regulation is believed to be fundamen- tally important in the control of CDC2 kinase function, i.e. the activity of ~ 3 4 ~ ~ ~ ' is regulated by complex formation with cyclins (Draetta et al, 1989) and phosphorylation of tyrosine-15 and threonine-161 (Gould & Nurse, 1989). However, recent evidence suggests that only a newly synthesized CDC2 protein can be modified and activated to Correspondence: Dr Masaki Saito, Division of Haemopoiesis, Institute of Haematology, Jichi Medical School. Minamikawachi- machi. Kawachi-gun. Tochigi 329-04, Japan. exhibit its kinase activity (McGowan et al, 1990; Welch zy 81 Wang, 1992). Therefore regulation at the transcriptional level might also be important for control of CDC2 kinase activity. There is increasing evidence that p34cdc2 levels in mammalian cells are modulated in response to cellular growth states by regulating levels of cdc2 mRNA. in contrast to earlier observations in yeast (Durkacz et al, 1986). Cdc2 mRNA is almost undetectable in resting cells such as peripheral blood T-lymphocytes or serum-starved fibroblasts, and it increases in a regulatory manner as cells enter the cell cycle by mitogenic stimuli (Lee et al, 1988: Furukawa et al, 1990). In contrast, cdc2 mRNA transcript is constitutively expressed in certain subsets of cancer cells or cell lines of which substantial numbers are in a cycling state, suggesting that deregulated expression of CDC2 might play a role in altered growth of these cells (Keyomarsi & Pardee, 1993). Recently,we have reported that cdc2 mRNA expression is important for the proliferation of normal haemopoietic cells and its down-regulation is closely associated with their differentiation (Furukawa et al, 1990. 1994). Therefore it is reasonable to speculate that abnormalities of the cdc2 gene are involved in the development of leukaemia or in the altered growth of leukaemic cells. To test this possibility, we investigated the expression of cdc2 mRNA transcripts by 94 zyxwvuts 0 1995 Blackwell Science Ltd