Byeong-Chel Lee

SA-1, a nuclear protein encoded by one member of a novel gene family: molecular cloning and detection in hemopoietic organs.

We report the molecular cloning of a novel gene family. The first member of this family was cloned from a mouse lambda gt11 cDNA library using the B92 monoclonal antibody (mAb) raised against stromal cell extracts. This was followed by RACE-PCR using mRNA from the stromal cell line. A 4 kb cDNA was obtained encoding a unique protein sequence of 1258 aa, that we designate stromal antigen (SA)-1. The human SA-1 gene was cloned by homology from a thymus cDNA library and the sequence of the predicted protein was found to be highly homologous to the murine SA-1 (>98.9%). Another cDNA was cloned and the deduced protein (SA-2) was 71% homologous to SA-1. Northern blot and PCR analysis indicated that on the mRNA level the SA-1 gene is expressed in all tissues analyzed and probably encodes a single transcript. The identification of SA-1 protein in tissues and cells required combined immunoprecipitation and Western blotting using a polyclonal antiserum raised against a predicted peptide of SA-1 and the B92 mAb. Using this assay we identified a protein of about 120 kDa in hemopoietic organs. Subcellular fractionation indicated that SA-1 is a nuclear protein. Thus, despite the ubiquitous expression on the mRNA level, the protein was predominantly detected in hemopoietic organs and may therefore be controlled on a post-transcriptional level. The SA-1 gene described in this study is highly conserved between mouse and man. This implies a crucial function for this protein.

Carboxyl-Terminal Src Kinase Homologous Kinase Negatively Regulates the Chemokine Receptor CXCR4 through YY1 and Impairs CXCR4/CXCL12 (SDF-1α)–Mediated Breast Cancer Cell Migration

Using microarray gene analysis, we found that carboxyl-terminal Src kinase homologous kinase (CHK) regulated the expression of the chemokine receptor, CXCR4. Northern blot and fluorescence-activated cell-sorting analyses showed that CHK down-regulated CXCR4 mRNA and protein levels, respectively. Mutated CHK, which contains a mutation within the ATP binding site of CHK, failed to inhibit CXCR4 expression, thus suggesting that CHK kinase activity is involved in the regulation of CXCR4. Results from gel shift analysis indicated that CHK regulates CXCR4 transcriptional activity by altering YY1 binding to the CXCR4 promoter. Whereas CHK had no significant effects on the expression of YY1, c-Myc, Max, and other YY1-binding proteins, CHK was found to modulate the YY1/c-Myc association. Furthermore, CHK inhibited CXCR4-positive breast cancer cell migration. Taken together, these studies show a novel mechanism by which CHK down-regulates CXCR4 through the YY1 transcription factor, leading to decreased CXCR4-mediated breast cancer cell motility and migration.

Reorganization of nuclear factors during myeloid differentiation

Differentiation in several stem cell systems is associated with major morphological changes in global nuclear shape. We studied the fate of inner‐nuclear structures, splicing factor‐rich foci and Cajal (coiled) bodies in differentiating hemopoietic, testis and skin tissues. Using antibodies to the splicing factors PSF, U2AF65 and snRNPs we find that these proteins localize in foci throughout the nuclei of immature bone marrow cells. Yet, when granulocytic cells differentiate and their nuclei condense and become segmented, the staining localizes in a unique compact and thread‐like structure. The splicing factor‐rich foci concentrate in the interior of these nuclei while the nuclear periphery and areas of highly compact chromatin remain devoid of these molecules. Differentiated myeloid cells do not stain for p80 coilin, the marker for Cajal bodies. Immature myeloid cells contain Cajal bodies although these usually do not coloclaize with PSF‐rich foci. Following complete inhibition of transcription in myeloid cells, the threaded PSF pattern becomes localized in several foci in the different lobes of mature granulocytes while in human HL‐60 immature myeloid leukemia cells PSF is found in the perinucleolar compartment. Studies of other differentiating stem cell systems show that PSF staining disappears completely in differentiated, transcriptionally inactive sperm cells, is scarce as cells migrate from the inner skin layers outward and is lost as cells of the hair follicle mature. We conclude that the formation and distribution of splicing factor‐rich foci in the nucleus during differentiation of various cell lineages is dependent on the levels of chromatin condensation and the differentiation status of the cell. J. Cell. Biochem. 81:379–392, 2001.

Enhanced proteolysis of pre-mRNA splicing factors in myeloid cells

OBJECTIVE Molecular identification and characterization of the bone marrow nuclear protein detected by the B92 monoclonal antibody. MATERIALS AND METHODS The protein was purified to homogeneity from acute myeloid leukemia cells and was subjected to peptide digestion and amino acid sequencing. Identified sequences were used to screen a bone marrow cDNA library in search of matching transcripts. The protein was further studied in different cells and tissues by examination of protease inhibitors and harsh lytic conditions and during apoptosis in HL-60 cells. RESULTS We found that the apparent bone marrow specific protein is a 47 kD proteolytic cleavage product of PSF, an essential pre-mRNA splicing factor. PSF is completely cleaved to p47 during lysis of immature myeloid cells due to potent proteolytic activity found in these cells but is rare in other cells and tissues. Furthermore, p47 is abundant in intact normal and tumor myeloid cells while in other cell types it is undetectable. The cleavage of PSF is accompanied by digestion of the PTB splicing regulator but not other proteins tested. In contrast, during apoptosis PTB is degraded while PSF remains intact. CONCLUSIONS The bone marrow 47 kD protein is a fragment constituting the N-terminal, protease-resistant half of the splicing factor PSF. Proteolytic degradation of PSF specifically occurs in intact myeloid cells and this process is enhanced upon myeloid cell lysis.

Synergistic Interactions between Heregulin and Peroxisome Proliferator-activated Receptor-γ (PPARγ) Agonist in Breast Cancer Cells

Here, we demonstrate that troglitazone (Rezulin), a peroxisome proliferator-activated receptor agonist, acted in synergy with heregulin to induce massive cell death in breast cancer cells. Although the combination of heregulin and troglitazone (HRG/TGZ) induced both apoptosis and necrosis, the main mode of cell death was caspase-independent and occurred via necrosis. This combination increased generation of superoxide in mitochondria, which in turn destabilized mitochondria potential. Pretreatment with N-acetyl-l-cysteine and catalase expression ameliorated cell death induced by the combination treatment, indicating a role of oxidative stress in mediating HRG/TGZ-induced cell death. Notably, pretreatment with pyruvate significantly prevented the cell death, suggesting a potential mechanistic link between metabolic stress and HRG/TGZ-induced cell death. The activation of the HRG signaling axis has been considered as a poor prognostic factor in breast cancer and confers resistance to gefitinib (Iressa) and tamoxifen. However, our data presented here paradoxically suggest that HRG expression can actually be beneficial when it comes to treating breast cancer with peroxisome proliferator-activated receptor-γ ligands. Taken together, the combination of HRG and TGZ may provide a basis for the development of a novel strategy in the treatment of apoptosis-resistant and/or hormone-refractory breast cancer.