JunB is a component of the Jun family genes of the activating protein-1 transcription factors that are important in the control of cell growth and differentiation and neoplastic transformation. Recently, it was demonstrated that transgenic mice specifically lacking JunB expression in the myeloid lineage developed a myeloproliferative disease, eventually progressing to blast crisis that resembled human chronic myeloid leukemia (CML). To gain further insights into the role of JunB in human CML, we examined peripheral blood from 17 healthy individuals and CML patients (11 in blastic crisis and 21 in chronic phase) by real-time quantitative reverse transcription-polymerase chain reaction analysis for the expression of JunB. The results showed the expression levels of JunB were significantly impaired in CML cases (blastic crisis < chronic phase < normal). Mutational analysis of the whole gene and methylation analysis of cytosine-phosphate guanosine (CpG) sites at the promoter area were further performed to investigate the possible mechanisms. However, no mutation was found within the coding region or the 9 flanking evolutionarily conserved regions in all CML cases. Interestingly, in the promoter area of JunB gene, most of the CpG sites were methylated in CML cases; in contrast, none of these CpG sites were methylated in normal cases. Demethylation by treatment of hypermethylated K562 cells with 5-aza-2'-deoxycytidine resulted in partial reactivation of JunB expression. Our results suggest that the down-regulated JunB expression in CML was due to the inactivation of JunB gene by methylation and the differential expression was correlated to the ratio of cells being methylated.

Summary. The expression of the suppressor of cytokine signalling-1 (SOCS1) protein is induced in response to stimulation by several cytokines. The induced SOCS1 inhibits the signalling pathway through the association with a variety of tyrosine kinase proteins. In this study, the mutation analyses, CpG island methylation status, and the expression of the SOCS1 gene in 112 chronic myeloid leukaemia (CML) samples, five leukaemia cell lines, and 30 normal controls were analysed. No genetic mutations of SOCS1 gene were noted in the CML samples. The SOCS1 gene was hypermethylated in 67% and 46% of the blastic and chronic phase CML samples respectively (P < 0AE0001). However, there was no methylation of the SOCS1 gene in normal controls or CML in molecular remission. The methylation status of the SOCS1 gene is consistent with the results of the real-time quantitative reverse transcription polymerase chain reaction and immunocytochemistry staining. Our results demonstrate that the SOCS1 gene silencing is caused by the methylation of CpG islands in CML and is reversed to an unmethylated status in molecular remission. As SOCS1 has universal activity to negatively regulate several cytokine signalling pathways, the loss of the negative regulation of cytokine signalling by the SOCS1 may play a role in the pathogenesis of CML progression.Keywords: SOCS1 gene, chronic myeloid leukaemia, methylation-specific PCR.The suppressor of cytokine signalling (SOCS) proteins are a family of negative regulators of cytokine signalling (Yoshimura et al, 1995;Endo et al, 1997;Naka et al, 1997;Starr et al, 1997;Krebs & Hilton, 2000. These proteins are relatively small molecules containing src homology 2 (SH2) domains. The SOCS proteins have been implicated in the negative regulation of several cytokine pathways, including the inhibition of the Janus kinase (JAK), tyrosine phosphorylation and nuclear translocation of the signal transducers and activators of transcription (STAT) proteins (Endo et al, 1997;Naka et al, 1997;Song & Shuai, 1998;Krebs & Hilton, 2000, interferon signalling (Song & Shuai, 1998;Alexander et al, 1999) and the suppression of steel factordependent proliferation (Sepulveda et al, 1999). The expression of the SOCS proteins is induced in response to stimulation by these cytokines, and the induced SOCS inhibits the signalling pathway (Krebs & Hilton, 2000. Among eight SOCS proteins [SOCS1-7 and cytokine inducible SH2 (CIS)-containing protein], the most potent inhibitors of cytokine signalling are SOCS1 and SOCS3 (Nicholson et al, 1999). The SOCS1 gene is located on chromosome 16p12-p13AE1 (Yandava et al, 1999). The genomic DNA contains two exons (Genbank accession number XP 008004), where the single-coding-exon transcribes a 1215-bp mRNA, which in turn encodes 211 amino acids. The SOCS1 transcript is often present in cells at low levels, and can be induced rapidly by a wide variety of cytokines, hormones, and growth factors. The SOCS1 protein inhibits the signalling of many cytokines including leukaemia inhibitory factor (LIF...

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