Abstract
An increasing number of Rho GTPase family proteins have been demonstrated to play critical roles in blood and immune cell development and function. The newly defined RhoH gene has been previously demonstrated to be mutated in lymphoma samples (Dallery et al, 1995; Pasqualucci et al, 2001). These alterations include chromosomal rearrangements and a high frequency of somatic mutations (up to 46%) in human non-Hodgkin’s lymphomas and diffuse large B-cell lymphoma. The RhoH gene encodes a novel hematopoietic-specific member of the RhoE subfamily, which is GTPase deficient, remaining in the active, GTP-bound state. Thus the activity of RhoH is likely regulated by the level of the protein expressed in the cell. The somatic mutations in the RhoH gene have been mapped to a 1.6kb hypermutable region in the intron 1, suggesting the possibility of dysregulated RhoH expression. However, levels of RhoH expression have not been directly measured in these hematopoietic tumors and so it remains unclear whether these mutations translate into aberrant RhoH expression. We utilized quantitative real-time RT-PCR to measure RhoH transcript levels in primary DLBCL patient samples. Based on morphologic and immunophenotypic analysis, 17 DLBCL positive samples and 14 normal control samples were used for our study. The levels of TATA-box binding protein (TBP) and human phosphogycerate kinase (HPGK) cDNAs were also examined simultaneously for relative expression normalization. RhoH transcript levels in a subset of the DLBCL samples were markedly reduced. In particular, 6 of 17 (~35%) tested samples showed a greater than 3-fold reduction in RhoH expression based on both RhoH/TBP and RhoH/HPGK ratios when compared with the median RhoH expression level of 14 normal samples. Overall, RhoH expression levels of the DLBCL group were significantly altered (mainly decreased) as compared with those of the normal group (p < 0.04, student T-test). To further determine correlation of the abnormal RhoH expression with somatic mutations in the hypermutable region of the RhoH gene in the DLBCL samples, we performed genomic PCR amplification and sequencing analysis of this region from the normal and DLBCL samples. In addition, we utilized a computational approach (Trafac - http://trafac.cchmc.org) to identify evolutionarily conserved putative transcription factor binding sites (TFBS) between human and other species in the hypermutable region. 13 conserved TFBS between human and mouse were identified in the hypermutable region. Mutations in the DLBCL patients are localized in 6 of these predicted TFBS, including pancreatic and duodenal homeobox 1 (PDX1), zinc-finger binding protein-89 (ZBP-89), lymphoid enhancer factor 1 (LEF-1), BRIGHT, engrailed 1 and myelin transcription factor 1 (MyT1). Interestingly, LEF-1 and BRIGHT are B cell-specific transcription activators. These results suggest that RhoH expression is frequently altered in 35–40% of DLBCL samples and mutations in the hypermutable region of the RhoH gene in several cases encompass core binding sequences of transcription factors important in B cell development.
PMA/IONO affects diffuse large B-cell lymphoma cell growth through upregulation of A20 expression