Search

The CCAAT/enhancer-binding protein alpha (C/EBP alpha) is essential for maintaining the differentiated state of hepatocytes in vivo. C/EBP alpha activates albumin transcription and coordinates the expression of multiple ornithine cycle enzymes involved in urea production. We have examined the effects of the C/EBP alpha knock-in gene on the mRNA and protein expression of genes involved in liver-specific functions, such as albumin and urea production. Albumin mRNA and protein levels are higher in knock-in hepatocytes compared with wild-type hepatocytes. mRNA levels for the ornithine cycle enzymes, namely arginase, carbamylphosphate synthetase 1, and ornithine transcarbamylase, also increase. Albumin secretion and urea production are sustained at higher levels in culture in knock-in hepatocytes. We have previously established that C/EBP alpha induces early liver glycogen storage in C/EBP alpha knock-in mice. Our present observations underline the importance of C/EBP alpha in liver metabolism and suggest that the induction of C/EBP alpha expression enhances hepatocyte function.

Hypoxia-inducible factor 1 (HIF-1) is the central mediator of cellular responses to low oxygen and has recently become an important therapeutic target for solid tumor therapy. Inhibition of HIF-1 is expected to result in the attenuation of hypoxia-inducible genes, which are vital to many aspects of tumor biology, including adaptative responses for survival under anaerobic conditions. To identify small molecules inhibiting the HIF-1 pathway, we did a biological screen on a 10,000-membered natural product-like combinatorial library. The compounds of the library, which share a 2,2-dimethylbenzopyran structural motif, were tested for their ability to inhibit the hypoxic activation of an alkaline phosphatase reporter gene under the control of hypoxiaresponsive elements in human glioma cells. This effort led to the discovery of 103D5R, a novel small-molecule inhibitor of HIF-1a. 103D5R markedly decreased HIF-1a protein levels induced by hypoxia or cobaltous ions in a dose- and timedependent manner, whereas minimally affecting global cellular protein expression levels, including that of control proteins such as HIF-1h ,I nBaa, and hh-actin. The inhibitory activity of 103D5R against HIF-1a was clearly shown under normoxia and hypoxia in cells derived from different cancer types, including glioma, prostate, and breast cancers. This inhibition prevented the activation of HIF-1 target genes under hypoxia such as vascular endothelial growth factor (VEGF) and glucose transporter-1 (Glut-1). Investigations into the molecular mechanism showed that 103D5R strongly reduced HIF-1a protein synthesis, whereas HIF-1a mRNA levels and HIF-1a degradation were not affected. 103D5R inhibited the phosphorylation of Akt, Erk1/2, and stressactivated protein kinase/c-jun-NH2-kinase, without changing the total levels of these proteins. Further studies on the mechanism of action of 103D5R will likely provide new insights into its validity/applicability for the pharmacologic targeting of HIF-1aa for therapeutic purposes. (Cancer Res 2005; 65(2): 605-12)

The CCAAT/enhancer binding protein alpha (C/EBP alpha) binds to specific promoter sequences and directs transcription of many genes expressed in the liver. Overexpression of C/EBP alpha in established cell lines inhibits cell proliferation. Primary hepatocytes from newborn C/EBP alpha(-/-) mice and normal littermates were used to determine whether the absence of C/EBP alpha increased proliferation and/or transformation of these cells in vitro. DNA synthesis, as measured by bromodeoxyuridine (BrdU) incorporation 24 hours postharvest, was fourfold higher in cells from C/EBP alpha(-/-) pups. Established cell lines were derived from 7 of 8 hepatocyte cultures initiated from null mutants, 4 of 23 cultures from heterozygotes, and 0 of 12 cultures from wild-type animals. C/EBP alpha(-/-) cultures had epithelial morphology, showed bile canaliculi, and expressed albumin messenger RNA (mRNA). When cultured on Matrigel, which promotes differentiation, cell lines derived from C/EBP alpha(-/-) mice formed cords and increased albumin mRNA expression by 1.7- to 3.8-fold. C/EBP alpha(-/-) cell lines exhibited rapid growth and rapid accumulation of chromosomal abnormalities, and were capable of forming nodules when inoculated into the abdominal subcutaneous tissue of nude mice. Our data show that C/EBP alpha is an important regulator of hepatocyte proliferation and participates in the maintenance of the nontransformed hepatic phenotype in vitro.

Transcription factors are master regulatory switches of differentiation, including the development of specific hematopoietic lineages from stem cells. Here we show that mice with targeted disruption of the CCAAT enhancer binding protein α gene (C/EBPα) demonstrate a selective block in differentiation of neutrophils. Mature neutrophils and eosinophils are not observed in the blood or fetal liver of mutant animals, while other hematopoietic lineages, including monocytes, are not affected. Instead, most of the white cells in the peripheral blood of mutant mice had the appearance of myeloid blasts. We also observed a selective loss of expression of a critical gene target of CCAAT enhancer binding protein α, the granulocyte colony-stimulating factor receptor. As a result, multipotential myeloid progenitors from the mutant fetal liver are unable to respond to granulocyte colony-stimulating factor signaling, although they are capable of forming granulocyte–macrophage and macrophage colonies in methylcellulose in response to other growth factors. Finally, we demonstrate that the lack of granulocyte development results from a defect intrinsic to the hematopoietic system; transplanted fetal liver from mutant mice can reconstitute lymphoid but not neutrophilic cells in irradiated recipients. These studies suggest a model by which transcription factors can direct the differentiation of multipotential precursors through activation of expression of a specific growth factor receptor, allowing proliferation and differentiation in response to a specific extracellular signal. In addition, thec/ebpα−/−mice may be useful in understanding the mechanisms involved in acute myelogenous leukemia, in which a block in differentiation of myeloid precursors is a key feature of the disease.

Mice homozygous for the targeted deletion of the c/ebp alpha gene, which expresses the CCAAT/enhancer-binding protein alpha (C/EBP alpha), did not store hepatic glycogen and died from hypoglycemia within 8 hours after birth. In these mutant mice, the amounts of glycogen synthase messenger RNA were 50 to 70 percent of normal and the transcriptional induction of the genes for two gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, was delayed. The hepatocytes and adipocytes of the mutant mice failed to accumulate lipid and the expression of the gene for uncoupling protein, the defining marker of brown adipose tissue, was reduced. This study demonstrates that C/EBP alpha is critical for the establishment and maintenance of energy homeostasis in neonates.

Using the human/hamster cell line UCTP2A-3, we have generated and isolated three hybrids, each containing a novel human chromosome 3p break. All chromosome 3 materials distal to the breaks were lost. Two of the breakpoints were produced using aphidicolin induction; the third breakpoint occurred spontaneously. The aphidicolin-induced breaks were localized to 3p21.1 in hybrid AR1 and to p14.1 in hybrid AR2. The spontaneous break was localized to 3p11 in hybrid 2A-3-1. These hybrids were used to sublocalize 18 chromosome 3 probes further to five regions within 3cen-p21.1. The new hybrids and sublocalized markers will be useful in the study and characterization of the 3p11, 3p14.1, and 3p21.1 segments of chromosome 3.

A total of 5700 human chromosome 3-specific cosmid clones was isolated from a series of cosmid libraries constructed from somatic cell hybrids whose only human component was an entire chromosome 3 or a chromosome 3 containing an interstitial deletion removing 50% of long arm sequences. Several unique sequence chromosome 3-specific hybridization probes were isolated from each of 616 of these cosmids. These probes were then used to localize the cosmids by hybridization to a somatic cell hybrid deletion mapping panel capable of resolving chromosome 3 into nine distinct subregions. All 616 of the cosmids were localized to either the long or short arm of chromosome 3 and 63% of the short arm cosmids were more precisely localized. We have identified a total of 87 cosmids that contain fragments that are evolutionarily conserved. Fragments from these cosmids should prove useful in the identification of new chromosome 3-specific genes as well as in comparative mapping studies. The localized cosmids should provide excellent saturation of human chromosome 3 and facilitate the construction of physical and genetic linkage maps to identify various disease loci including Von Hippel Lindau disease and renal and small cell lung carcinoma.

FAM-labeled oligo dT (FAMdT) was utilized as a means to gauge efficient transfection of small nucleic acids into mouse embryonic stem cell (mESC) colonies. Using colonies grown overnight, transfection was restricted largely to the periphery of the colonies with only a 40% decrease in Oct-3/4 RNA transcript levels following cognate Oct-3/4, small interfering RNA (siRNA) delivery. However, transfection of mESC 4 h after seeding gave greater than 90% cells being successfully transfected based on quantitative real-time PCR detection of approximately 90% Oct-3/4 RNA transcript knockdown. This method provides an economical and efficient means by which to determine effective transfection conditions, and establish efficient siRNA knockdown of reportedly difficult to transfect cell lines such as mESC.

Gene amplification is frequently present in human tumors, although specific target genes relevant to many amplified loci remain unidentified. An expression cloning assay enabled identification of a candidate oncogene derived from human chromosome 3p14.1. The cDNA retrieved from morphologically transformed cells contained the full-length protein coding region and detected an abundant transcript in the same cells. Sequence analysis revealed identity with the wild-type sequence of p44S10, a highly conserved subunit of the 26S proteasome that exhibits similarity to the Arabidopsis fus6/cop11 family of signaling molecules. p44S10 gene copy number and mRNA expression were increased in association with segmental 1.8 - 11-fold chromosomal gains in cutaneous malignant melanoma cell lines (5/13; 40%) and tumors (2/40; 5%), and in breast cancer MCF-7 cells. Likewise, malignant progression of human radial growth phase WM35 melanoma cells was associated with amplification and increased expression of endogenous p44S10, and increased expression of p44S10 was sufficient to induce proliferation of WM35 cells in vivo. The results demonstrate segmental copy number gains within chromosome 3p in cutaneous malignant melanoma and suggest that deregulation of a proteasome regulatory particle subunit may contribute to the malignant phenotype.

The constitutive 3p14.2 fragile site is the most highly inducible fragile site in the human genome. This locus may predispose chromosome 3 to specific losses due to the deletions and translocations that have been associated with several malignancies, including hereditary renal cell carcinoma. Using aphidicolin concentrations of 0.4 and 4.0 [mu]M, the authors have generated and isolated 23 and 22 respective somatic cell hybrids that contain chromosome 3 short-arm breaks. The breakpoints in these hybrids have been localized with numerous chromosome 3 markers. They have observed that at the low aphidicolin dose, chromosome-3 breaks cluster within the 3q14.2 region, whereas at the high aphidicolin dose, two new loci, one within 3p14.1 and the other near the centromere, become predominantly affected. The studies have failed to differentiate any of the 3p14.2 breakpoints from each other or from the t(3;8)(p14.2;q24.13) familial renal cell carcinoma translocation breakpoint, suggesting that the fragile site may have played a role in the generation of this balanced translocation. The resulting somatic cell hybrids generated from this work have refined the marker localizations within 3q13-p21.1 and should facilitate the physical characterization of this interesting region. 32 refs., 4 figs., 2 tabs.