CBFb-SMMHC is expressed from the inv(16) chromosome in M4Eo AML. Mice lacking CBF subunits or expressing the CBFb-SMMHC or AML1-ETO oncoproteins failed to develop de®nitive hematopoiesis. To investigate these e ects on hematopoiesis, we expressed CBFb-SMMHC from the metallothionein promoter, in both 32D cl3 myeloid cells and Ba/F3 B-lymphoid cells. Addition of zinc increased CBFb-SMMHC levels more than tenfold, with higher levels evident in Ba/F3 lines. Levels obtained in 32D cl3 cells were similar to those of endogenous CBFb. Indirect immuno¯uorescence revealed zinc-inducible speckled, nuclear staining in Ba/F3 cells and di use nuclear staining in 32D cl3 cells. CBFb-SMMHC reduced endogenous CBF DNA-binding ®vefold in both cell types, increased cell generation time 1.9-fold, on average, in 32D cl3 cells and 1.5-fold in Ba/ F3 cells and decreased tritiated thymidine incorporation into DNA correspondingly. CBFb-SMMHC increased the proportion of cells in G1 1.7-fold, on average, in 32D cl3 and Ba/F3 cells, and decreased the proportion of cells in S phase by a similar degree. CBFb-SMMHC induced a marked increase in hypophosphorylated Rb, but did not alter IL-3 Receptor a or b subunit levels. Neither apoptosis nor 32D di erentiation was induced by zinc in IL-3 in these lines. Induction of CBFb-SMMHC in 32D cl3 cells did not inhibit their di erentiation to neutrophils or their expression of myeloperoxidase mRNA in G-CSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentiate inhibition of di erentiation by CBFb-SMMHC by allowing its increased expression.
To identify the biochemical events which specify early myeloid differentiation, we have investigated the regulation of the murine myeloperoxidase (MPO) gene. Our initial functional dissection of the MPO promoter region in differentiating 32D cl3 myeloid cells delimited a 414-bp proximal MPO enhancer and identified several functional cis elements within this region (39) (Fig. 1A). One of these cis elements, 5Ј-AACCACA-3Ј, was critical for enhancer function and bound a factor that we subsequently identified as core binding factor (CBF) (24). The CBF␣ subunits are encoded by a family of three genes. Each contains an N-terminal DNA-binding runt domain (16,19,28) and a C-terminal transactivating domain (1,20). The CBF subunit interacts with the CBF␣ subunits via the runt domain and increases their affinities for DNA (29,43). Mice null for either the CBF␣2 (AML1) gene or the CBF gene died in utero and lacked all lineages of definitive hematopoiesis (30,41,42). In our initial study, we found that mutation of the CBF-binding site at Ϫ288 in the proximal MPO enhancer decreased enhancer activity 30-fold. However, one or two CBF-binding sites activated a minimal thymidine kinase (TK) promoter only threefold (39).We devised a novel deletional strategy to identify all of the cis elements within the MPO proximal enhancer required for cooperation with CBF in 32D cl3 cells. The CBF-binding site was linked first to a 5Ј enhancer deletion series and then to a 3Ј deletion series which retained the minimal downstream region not required for synergy. CBF strongly activated the MPO enhancer only when c-Myb was bound to one of two conserved c-Myb-binding sites, and optimally when c-Myb bound both sites. These c-Myb-binding sites are separated by 154 bp. An evolutionarily conserved GA-rich element adjacent to the downstream c-Myb site (Fig. 1C) contributed to cooperativity when a c-Myb-binding site was present. Our deletional strategy should be generally applicable to efforts to identify the most important interactions between multiple factors shown to regulate a particular enhancer.CBF and c-Myb were shown to synergistically regulate the T-cell receptor ␦-chain (TCR␦) gene in T-lymphoid cells (13), but the MPO gene is the first gene found to be activated synergistically by these factors in myeloid cells. Both CBF and c-Myb contributed to increased activity of the MPO proximal enhancer during 32D cl3 cell differentiation. A similar role for these factors during lymphoid maturation has not been demonstrated. In the TCR␦ enhancer, cooperation depended on a critical spacing between the CBF-and c-Myb-binding sites (13). This requirement was not evident for the MPO enhancer in 32D cl3 myeloid cells, perhaps reflecting the involvement of different CBF family members or a different mechanism of synergy.CBF and c-Myb are both myeloid proto-oncoproteins (10). These factors may together regulate genes critical for myeloid proliferation, and determining how CBF and c-Myb cooperate in myeloid cells might contribute to our understanding of leukem...
We have expressed several variants of core binding factor  (CBF)-smooth muscle myosin heavy chain (SMMHC) from the metallothionein promoter in Ba/F3 cells. Deletion of amino acids 2-11 from the CBF segment, required for interaction with CBF␣, prevented CBF-SMMHC from inhibiting CBF DNA binding and cell cycle progression. Deletion of 283 carboxyl-terminal residues from the SMMHC domain, required for multimerization, also inactivated CBF-SMMHC. Nuclear expression of CBF(⌬2-11)-SMMHC was decreased relative to CBF-SMMHC. CBF(⌬2-11)-SMMHC linked to a nuclear localization signal still did not slow cell growth. The ability of each CBF-SMMHC variant to inhibit CBF DNA binding and cell proliferation correlated with its ability to inhibit transactivation by an AML1-VP16 fusion protein. Thus, CBF-SMMHC slows cell cycle progression from G 1 to S phase by inhibiting CBF DNA binding and transactivation.The core binding factor (CBF) 1 family is made up of transcription factors that contain a common CBF subunit and one of three CBF␣ subunits: CBF␣1, AML1 (CBF␣2), or CBF␣3 (1-7). CBF increases the affinity of the CBF␣ subunits for DNA, but does not bind DNA directly (4, 7). The CBF␣ subunits contain a domain required for heterodimerization and DNA binding (3,8).Translocations involving subunits of CBF are common in acute leukemias (9). Inv(16) is present in 10% of acute myeloid leukemias (AMLs) and encodes CBF-SMMHC, in which CBF is fused to the tail domain of SMMHC (10). t(8;21) is present in 12% of AMLs and encodes AML1-ETO, which includes the DNA-binding domain of AML1 (11). 25% of pediatric B-lineage acute lymphocytic leukemias contain t(12;21), which encodes TEL-AML1 (12, 13).Each of these "CBF oncoproteins" inhibits CBF activities (14 -20). Also, mice expressing CBF-SMMHC or AML1-ETO fail to develop definitive hematopoiesis, just as do mice lacking AML1 or CBF (21-26).CBF activates the expression of several lymphoid and myeloid genes, suggesting that lack of differentiation accounts for the phenotypes of CBF null mice (27-30). We expressed CBF-SMMHC from the zinc-responsive MT promoter in 32D cl3 myeloid and Ba/F3 B-lymphoid cells (14). Induction of CBF-SMMHC resulted in decreased CBF DNA binding and slowed proliferation during G 1 phase. The differentiation of 32D cl3 cells in response to granulocyte colony-stimulating factor was unaffected. We proposed that initial genetic alterations occur during leukemogenesis that bypass the growth inhibitory effect of CBF-SMMHC, potentiating inhibition of differentiation.We have now expressed several CBF-SMMHC mutants in Ba/F3 cells. Amino acids 1-165 of the 182-amino acid CBF protein are present in the majority of CBF-SMMHC fusion proteins, although a variant containing only residues 1-133 is present in rare patients (31). Amino acids 1-73 and 102-137 are highly conserved between Drosophila Brother and Bigbrother and murine CBF (32). Segment 1-137 is sufficient to strongly increase the affinity of CBF␣ subunits for DNA, whereas segment 1-133 or 1-132 does so o...
Hoag JB, Liu M, Easley RB, Britos-Bray MF, Kesari P, Hassoun H, Haas M, Tuder RM, Rabb H, Simon BA. Effects of acid aspiration-induced acute lung injury on kidney function. Am J Physiol Renal Physiol 294: F900-F908, 2008. First published February 6, 2008 doi:10.1152/ajprenal.00357.2007.-Acute lung injury (ALI) in combination with acute kidney injury carries a mortality approaching 80% in the intensive care unit. Recently, attention has focused on the interaction of the lung and kidney in the setting of ALI and mechanical ventilation (MV). Small animal models of ALI and MV have demonstrated changes in inflammatory mediators, water channels, apoptosis, and function in the kidney early in the course of injury. The purpose of this investigation was to test the hypothesis that ALI and injurious MV cause early, measurable changes in kidney structure and function in a canine HCl aspiration model of ALI when hemodynamics and arterial blood gas tensions are carefully controlled. Intratracheal HCl induced profound ALI as demonstrated by increased shunt fraction and airway pressures compared with sham injury. Shaminjured animals had similar mean arterial pressure and arterial PCO 2 and HCO3 levels compared with injured animals. Measurements of renal function including renal blood flow, urine flow, serum creatinine, glomerular filtration rate, urine albumin-to-creatinine ratio, and kidney histology scores were not different between groups. With maintenance of hemodynamic parameters and alveolar ventilation, ALI and injurious MV do not alter kidney structure and function early in the course of injury in this acid aspiration canine model. Kidney injury in large animal models may be more similar to humans and may differ from results seen in small animal models. mechanical ventilation; biotrauma; renal function PATIENTS WITH COMBINED ACUTE lung injury (ALI) and acute kidney injury (AKI) have a mortality rate approaching 80% in the intensive care unit (17). Considerable clinical and experimental data support the existence of a direct pulmonary-renal interaction in the setting of ALI and the acute respiratory distress syndrome (ARDS). In the Acute Respiratory Distress Syndrome Network study (1) comparing low tidal volume to "conventional" tidal volume (V T ) ventilation, protective modes of ventilation not only improved mortality from ARDS, but led to improved function in other organ systems. Specifically, patients receiving 6 ml/kg V T had a lower number of days with renal failure in the first 28 days compared with patients receiving 12 ml/kg V T (1). Similarly, a smaller study (24) showed a decrease in the number of patients developing renal failure in the first 72 to 96 h when low tidal volume ventilation was used.Likewise, animal models of ALI and mechanical ventilation have been used in an effort to determine mechanisms of organ cross talk in response to injury. Most well-described are the influences of mechanical ventilation in the setting of ALI on hemodynamics, thus modifying renal blood flow. Positive end-expiratory pre...
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