In this study, we propose a new role of Helios in the BCR-mediated apoptosis and O2−-generating activity in immature B lymphocytes. Lymphocyte development requires numerous transcription factors [1], [2], [3], [4], [5], [6], [7] and [8]. Among them, Ikaros family proteins with zinc finger motif are essential for normal lymphoid cell differentiation and proliferation

[5], [6], [9] and [10]. For example, lack of Ikaros in the hematopoietic cells leads to complete block in differentiation selleck chemicals llc to both B and T lymphocytes [21]. In addition, their abnormalities cause several lymphoid malignancies such as leukemias and SP600125 nmr lymphomas [6], [11], [12] and [13]. Helios, one of the Ikaros family proteins, is preferentially expressed in T lymphocytes [9], [20], [21], [22] and [23]. Moreover, transgenic expression of Helios in B lymphocytes alters their properties and promotes lymphomagenesis, suggesting that silencing of Helios is critical for normal function of B lymphocytes [24]. In agreement with previous reports in B lymphocytes [9], [25] and [29], the steady-state level of Helios mRNA was very low in DT40 (see many PCR cycle

numbers in Fig. 1C). On the other hand, Ikaros family proteins are generally known to be dimerized with other family members or themselves [9]. The Helios gene is transcribed as various isoforms by alternative splicing in a similar manner as other Ikaros family proteins [9], [13] and [25]. The short forms lacking the zinc-finger motif can behave as dominant negative isoforms upon heterodimerization [9] and [13]. Since Helios is able to heterodimerize with Ikaros or Aiolos [9],

[20] and [22], we thought that Helios plays a role as a modulator of other Ikaros family proteins, in spite of its very low levels in B lymphocytes. However, little is known about the physiological function of Helios in B lymphocytes. Using gene targeting techniques in DT40, we could study the Staurosporine manufacturer physiological functions of various genes in immature B lymphocytes without the influences of other developmental stages, unlike knock-out mice. To clarify in vivo roles of Helios in immature B lymphocytes, we first generated homozygous DT40 mutant, Helios−/−, devoid of two alleles of the Helios gene ( Fig. 1). Aiolos-deficiency caused alterations in the expressions of several genes: bak, caspase-9, inhibitor of CAD and PKCs (PKC-α, PKC-β, PKC-δ, PKC-ε, PKC-η and PKC-ζ) [19]. In order to examine whether or not Helios participates in the expressions of these genes, we first carried out semiquantitative RT-PCR on total RNAs prepared from Helios−/−, Aiolos−/− and DT40.