Runx3/Pebp2alphaC null mouse gastric mucosa exhibits hyperplasias due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to growth-inhibitory and apoptosis-inducing action of TGF-beta, indicating that Runx3 is a major growth regulator of gastric epithelial cells. Between 45% and 60% of human gastric cancer cells do not significantly express RUNX3 due to hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of human gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain abolished the tumor-suppressive effect of RUNX3, suggesting that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.

T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4(+)CD8(+)), which are selected to become either CD4(+)CD8(-)helper cells or CD4(-)CD8(+) cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4(-)CD8(+) thymocytes. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4(-)CD8(-) thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes.

A murine transcription factor, PEBP2, is composed of two subunits, a and ,B. There are two genes in the mouse genome, PEBP2aA and PEBP2&aB, which encode the at subunit. Two types of the aLB cDNA clones, aLBi and aB2, were isolated from mouse fibroblasts and characterized. They were found to represent 3.8-and 7.9-kb transcripts, respectively. The 3.8-kb RNA encodes the previously described aB protein referred to as oiBl, while the 7.9-kb RNA encodes a 387-amino-acid protein, termed aB2, which is identical to aBl except that it has an internal deletion of 64 amino acid residues. Both otBl and aB2 associate with PEBP2,I and form a heterodimer. The atB2/4 complex binds to the PEBP2 binding site two-to threefold more strongly than the aB1/,I complex does. aBl stimulates transcription through the PEBP2 site about 40-fold, while aB2 is only about 25 to 45% as active as aBl. Transactivation domain is located downstream of the 128-amino-acid runt homology region, referred to as the Runt domain. Mouse chromosome mapping studies revealed that aA, aB, and ,B genes are mapped to chromosomes 17, 16, and 8, respectively. The last two genes are syntenic with the human AMLI on chromosome 21q22 and PEBP24/CBFD on 16q22 detected at the breakpoints of characteristic chromosome translocations of the two different subtypes of acute myeloid leukemia. These results suggest that the previously described chimeric gene products, AML1/MTG8(ETO) and AMLl-EAP generated by t(8;21) and t(3;21), respectively, lack the transactivation domain of AMLL.A murine transcription factor, PEBP2, also called PEA2, was identified as a factor which binds to the region of the polyomavirus enhancer originally termed the PEA2 site (35). PEBP2 functionally cooperates with other enhancer corebinding proteins, including AP1 and Ets family proteins, PEA3 (48), , and PEBP5 (2), and plays an important role in stimulation of transcription and viral DNA replication. PEBP2 is composed of a and 3 subunits (32, 34). The at subunit bind to DNA by recognizing the consensus sequence, (Pu/T) ACCPuCA (13,34). The companion 3 subunit binds to the cx protein and increases the affinity of the a protein to DNA without binding to DNA by itself (32). cDNAs coding for both subunits have been cloned and characterized in detail (32, 34).PEBP2 has recently been shown to be closely related to acute myeloid leukemia (AML). The t(8;21)(q22;q22) translocation is one of the most frequent chromosome abnormalities in AML and is classified into the FAB-M2 subtype of AML (39). It has been shown that the t(8;21) breakpoints in the chromosome 21 are clustered in the recently described AMLI gene (27), the product of which shares a high homology within the 128-amino-acid (aa) region with the product of the Drosophila segmentation gene, runt (14), and the at subunit of PEBP2 (8,9,34 genome, oaB, which shares a high sequence homology with the original ao gene, termed aA. The aB gene as a whole is highly homologous to human AMLl (3). The discovery of the homology of the AML1 protein to the otsubunit o...

RUNX3 is inactivated at high frequency in many tumors. However, in most cases, inactivation is caused by silencing of the gene due to promoter hypermethylation. Because epigenetic silencing is known to affect many major tumor suppressor genes in cancer cells, it is not clear whether RUNX3 is primarily responsible for the induction of carcinogenesis in these cases, except for the gastric cancer cases that we reported previously. We investigated genetic and epigenetic alterations of RUNX3 in 124 bladder tumor cases and seven bladder tumor-derived cell lines. Here we show that RUNX3 is inactivated by aberrant DNA methylation in 73% (90 of 124) of primary bladder tumor specimens and 86% (six of seven) of bladder tumor cell lines. In contrast, the promoter regions of 20 normal bladder mucosae were unmethylated. Importantly, one patient bore missense mutations, each of which resulted in amino acid substitutions in the highly conserved Runt domain. The mutations abolished the DNA-binding ability of RUNX3. A second patient had a single nucleotide deletion within the Runt domain coding region that resulted in truncation of the protein. RUNX3 methylation was a significant risk factor for bladder tumor development, superficial bladder tumor recurrence, and subsequent tumor progression. These results strongly suggest that inactivation of RUNX3 may contribute to bladder tumor development and that promoter methylation and silencing of RUNX3 could be useful prognostic markers for both bladder tumor recurrence and progression. (Cancer Res 2005; 65(20): 9347-54)

Each of the two human genes encoding the ␣ and ␤ subunits of a heterodimeric transcription factor, PEBP2, has been found at the breakpoints of two characteristic chromosome translocations associated with acute myeloid leukemia, suggesting that they are candidate proto-oncogenes. Polyclonal antibodies against the ␣ and ␤ subunits of PEBP2 were raised in rabbits and hamsters. Immunofluorescence labeling of NIH 3T3 cells transfected with PEBP2␣ and -␤ cDNAs revealed that the full-size ␣A1 and ␣B1 proteins, the products of two related but distinct genes, are located in the nucleus, while the ␤ subunit is localized to the cytoplasm. Deletion analysis demonstrated that there are two regions in ␣A1 responsible for nuclear accumulation of the protein: one mapped in the region between amino acids 221 and 513, and the other mapped in the Runt domain (amino acids 94 to 221) harboring the DNA-binding and the heterodimerizing activities. When the full-size ␣A1 and ␤ proteins are coexpressed in a single cell, the former is present in the nucleus and the latter still remains in the cytoplasm. However, the N-or C-terminally truncated ␣A1 proteins devoid of the region upstream or downstream of the Runt domain colocalized with the ␤ protein in the nucleus. In these cases, the ␤ protein appeared to be translocated into the nucleus passively by binding to ␣A1. The chimeric protein containing the ␤ protein at the N-terminal region generated as a result of the inversion of chromosome 16 colocalized with ␣A1 to the nucleus more readily than the normal ␤ protein. The implications of these results in relation to leukemogenesis are discussed.