Transcription factors play a key role in the development and differentiation of specific lineages from multipotential progenitors. Identification of these regulators and determining the mechanism of how they activate their target genes are important for understanding normal development of monocytes and macrophages and the pathogenesis of a common form of adult acute leukemia, in which the differentiation of monocytic cells is blocked. Our previous work has shown that the monocyte-specific expression of the macrophage colony-stimulating factor (M-CSF) receptor is regulated by three transcription factors interacting with critical regions of the M-CSF receptor promoter, including PU.1 and AML1. PU.1 is essential for myeloid cell development, while the AML1 gene is involved in several common leukemia-related chromosome translocations, although its role in hematopoiesis has not been fully identified. Along with AML1, a third factor, Mono A, interacts with a small region of the promoter which can function as a monocyte-specific enhancer when multimerized and linked to a heterologous basal promoter. Here, we demonstrate by electrophoretic mobility shift assays with monocytic nuclear extracts, COS-7 cell-transfected factors, and specific antibodies that the monocyte-enriched factor Mono A is CCAAT enhancer-binding protein (C/EBP). C/EBP has been shown previously to be an important transcription factor involved in hepatocyte and adipocyte differentiation; in hematopoietic cells, C/EBP is specifically expressed in myeloid cells. In vitro binding analysis reveals a physical interaction between C/EBP and AML1. Further transfection studies show that C/EBP and AML1 in concert with the AML1 heterodimer partner CBF␤ synergistically activate M-CSF receptor by more than 60-fold. These results demonstrate that C/EBP and AML1 are important factors for regulating a critical hematopoietic growth factor receptor, the M-CSF receptor, suggesting a mechanism of how the AML1 fusion protein could contribute to acute myeloid leukemia. Furthermore, they demonstrate physical and functional interactions between AML1 and C/EBP transcription factor family members.

BRCA2 mutations predispose carriers mainly to breast cancer. The vast majority of BRCA2 mutations are predicted to result in a truncated protein product. The smallest known cancer-associated deletion removes from the C terminus only 224 of the 3,418 residues constituting BRCA2, suggesting that these terminal amino acids are crucial for BRCA2 function. A series of green fluorescent protein (GFP)-tagged BRCA2 deletion mutants revealed that nuclear localization depends on two nuclear localization signals that reside within the final 156 residues of BRCA2. Consistent with this observation, an endogenous truncated BRCA2 mutant (6174delT) was found to be cytoplasmic. Together, these studies provide a simple explanation for why the vast majority of BRCA2 mutants are nonfunctional: they do not translocate into the nucleus. G erm-line mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 predispose carriers mostly to breast cancer, but also to other cancers (reviewed in ref. 1). BRCA1 and BRCA2 account for 50% and 30%, respectively (2), of the inherited cases of breast cancer, which account for about 5-10% of all cases of breast cancer (3). These genes are classified as tumor suppressors because most BRCA1-and BRCA2-linked tumors have undergone loss of heterozygosity (LOH) at these loci. They have further been classified as ''caretakers'' on the basis of their proposed genome integrity maintenance functions as well as the infrequency of linked sporadic tumors (4).Although the precise biochemical functions of the BRCA1 and BRCA2 gene products have yet to be determined, there is substantial evidence linking both of them to transcriptional control and DNA repair (reviewed in refs. 5 and 6), activities consistent with their nuclear localization (7-17). For BRCA2, we present evidence that its inability to be translocated to the nucleus may explain why the vast majority of BRCA2 mutations are nonfunctional. Since nearly all BRCA2 mutations are predicted to encode truncated proteins (18), we sought to determine the biochemical consequence of the C terminus. The smallest known cancer-associated deletion of BRCA2 is predicted to remove 224 amino acids (7% of the coding sequence) from its C terminus (19). These C-terminal amino acids appear to be critical because the nuclear localization signals (NLSs) of BRCA2 reside within this region. Using a series of green fluorescent protein (GFP)-tagged BRCA2 deletion mutants, we found that nuclear localization depends upon two NLSs that reside within the final 156 residues of BRCA2. Consistent with this observation, an endogenous cancer-associated truncated BRCA2 mutant (6174delT) was found to be cytoplasmic. Consequently, truncation mutant forms of BRCA2 are predicted to encode nucleus-excluded gene products, providing a simple explanation for why these mutants are nonfunctional: they do not translocate into the nucleus. Plasmid Constructions. Full-length human BRCA2 was modified to contain NotI and SalI restriction sites that permitted directional cloning into mammalian exp...

Background The prescence of circulating tumor cells (CTCs) in the peripheral blood of cancer patients and their frequency has been correlated with disease status. Methods In this study, CTCs were characterized by flow cytometry and fluorescence microscopy after immunomagnetic enrichment from 7.5‐ml blood samples collected from patients with prostate cancer in evacuated blood‐draw tubes that contained an anticoagulant and a preservative. Events were classified as tumor cell candidates if they expressed cytokeratin, lacked CD45, and stained with the nucleic acid dye 4,6‐diamidino‐2‐phenylindole. Results In the blood of prostate cancer patients, only few of these events were intact cells. Other CTC events appeared as damaged cells or cell fragments by microscopy. By flow cytometry, these events stained variably with 4,6‐diamidino‐2‐phenylindole and frequently expressed the apoptosis‐induced, caspase‐cleaved cytokeratin 18. Similar patterns of cell disintegration were observed when cells of the prostate line LNCaP were exposed to paclitaxel before spiking the cells into normal blood samples. Conclusions The different observed stages of tumor cell degradation or apoptosis varied greatly between patients and were not found in blood of normal donors. Enumeration of CTCs and identification of CTCs undergoing apoptosis may provide relevant information to evaluate the response to therapy in cancer patients. © 2004 Wiley‐Liss, Inc.