Nobuo Kondoh

Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis

BACKGROUND/AIMS We identified the glucose-regulated protein (grp) 78 as a transformation-associated gene in hepatocellular carcinoma (HCC). Grp78 is a molecular chaperone involved in the unfolded protein response, the expression of which can be regulated by the transcription factors ATF6 and XBP1. Thus, we investigated the regulatory mechanisms of the grp78 gene in liver malignancy. METHODS Expression of grp78, ATF6 and XBP1 was examined by Northern blot, RT-PCR, immunoblot and immunohistochemical analyses. A reporter assay of the grp78 promoter was also performed. RESULTS Elevation of grp78 and ATF6 mRNAs and the splicing of XBP1 mRNA, resulting in the activation of XBP1 product, occurred in HCC tissues with increased histological grading. Higher accumulation of the grp78 product in the cytoplasm, concomitantly with marked nuclear localization of the activated ATF6 product (p50ATF6), was observed in moderately to poorly differentiated HCC tissues. Cooperation between the distal DNA segment and the proximal endoplasmic reticulum stress response elements was essential for maximum transcription of the grp78 promoter in HCC cells. CONCLUSIONS The endoplasmic reticulum stress pathway mediated by ATF6 and by IRE1-XBP1 systems seems essential for the transformation-associated expression of the grp78 gene in HCCs.

Serial analysis of gene expression in HIV-1-infected T cell lines

The gene expression profile of the HIV‐1 infection state was analyzed in the human T cell line MOLT‐4. Using the serial analysis of gene expression (SAGE) method, a total of 142 603 SAGE tags were sequenced and identified, representing 43 581 unique mRNA species. Comparison of expression patterns revealed that 53 cellular genes were differentially expressed upon HIV‐1 infection. Northern blot and RT‐PCR analyses confirmed the altered expression of the genes in both MOLT‐4 and MT‐4 cells. Up‐regulated genes were mainly composed of transcription factors and genes related to T cell activation, whereas down‐regulated genes were comprised of mitochondrial proteins, actin‐related factors and translational factors. These findings indicate that persistent T cell activation, which may accelerate HIV‐1 replication, and the disruption of cellular housekeeping genes including those involved in anti‐apoptotic systems, may play an important role in HIV‐1‐induced pathogenesis.

The role of Ets family transcription factor PU.1 in hematopoietic cell differentiation, proliferation and apoptosis

The PU.1 gene encodes an Ets family transcription factor which controls expression of many B cell- and macrophage-specific genes. Expression of the gene is critical for development of lymphoid and myeloid cell lineages, since PU.1-deficient mice exhibit defects in the development of these cell lineages. The PU.1 gene is identical to the Spi-1 gene isolated from common proviral integration sites in Friend virus-induced murine erythroleukemia (MEL), and deregulated expression of the gene is believed to be an essential step of the disease. We recently demonstrated that overexpression of PU.1 inhibits erythroid differentiation of MEL cells induced with the differentiating agent DMSO. We also noticed unexpectedly that overexpression of PU.1 together with DMSO induces marked growth arrest and apoptosis in MEL cells, supporting the notion that some oncogenes induce growth inhibition and apoptosis rather than cell proliferation and transformation under specific circumstances as shown with the c-myc gene. In this review, the role of PU.1 in hematopoietic cell differentiation, proliferation and apoptosis is described and the possible molecular mechanisms of PU.1-induced effects in MEL cells are discussed.

Serial analysis of gene expression in a microglial cell line

We used the serial analysis of gene expression (SAGE) method to systematically analyze transcripts present in a microglial cell line. Over 10,000 SAGE tags were sequenced, and shown to represent 6,013 unique transcripts. Among the diverse transcripts that had not been previously detected in microglia were those for cytokines such as endothelial monocyte‐activating polypeptide I (EMAP I), and for cell surface antigens, including adhesion molecules such as CD9, CD53, CD107a, CD147, CD162 and mast cell high affinity IgE receptor. In addition, we detected transcripts that were characteristic of hematopoietic cells or mesodermal structures, such as E3 protein, A1, EN‐7, B94, and ufo. Furthermore, the profile contained a transcript, Hn1, that is important in hematopoietic cells and neurological development (Tang et al. Mamm Genome 8:695–696, 1997), suggesting the probable neural differentiation of microglia from the hematopoietic system in development. Messenger RNA expression of these genes was confirmed by RT‐PCR in primary cultures of microglia. Significantly, this is the first systematic profiling of the genes expressed in a microglial cell line. The identification and further characterization of the genes described here should provide potential new targets for the study of microglial biology. GLIA 28:265–271, 1999.

Identification and characterization of differentially expressed mRNAs in HIV type 1-infected human T cells.

We used a novel differential display (DD) technique to identify host factors involved in virus replication, pathogenesis, and host response in HIV-1-infected T cells. Thirteen cDNA fragments differentially expressed in HIV-1NL4-3-infected MT-4 cells prior to the occurrence of specific apoptotic cell death were sequenced and identified. Two of seven elevated genes were identical to HIV-1 sequences and the other five were MIP-1alpha, ACTE-III, CD11c, arginase I, and CCR5. The six downregulated genes included prothymosin-a, Jaw-1, proteasome subunit XAPC7, splicing factor 9G8, GA17 protein, and an unknown mRNA. Northern blot and RT-PCR analyses confirmed the altered gene expressions in MT-4 cells as well as in another T cell line, MOLT-4. We also revealed that the amount of MIP-1alpha in culture supernatant of HIV-1-infected cells was increased by more than 15-fold relative to control cells, and the expression of its receptor CCR5 was cooperatively upregulated on the surface of these cells. Furthermore, the upregulation of CD11c after HIV-1 infection was slightly inhibited by blocking the MIP-1alpha-mediated signal transduction. These results indicate that genes altered on HIV-1 infection may be mutually organized and play an important role in HIV-1-induced pathogenesis.

Transformation-associated gene regulation by ATF6α during hepatocarcinogenesis

We have previously reported that the endoplasmic reticulum (ER) stress‐regulated transmembrane transcription factor 6 α (ATF6α) is implicated in the pathogenesis of hepatocellular carcinomas (HCCs). In order to further identify genes that are regulated by ATF6α, the global gene expression profiles of the ATF6α‐transfected and untransfected HCC cell line, HLF, were analyzed. These results were then compared with the differential gene expression patterns of poorly differentiated HCC and control non‐tumorous liver tissue. Our findings demonstrate that at least 18 genes are specifically upregulated by ATF6α, while another UPR mediator, XBP1 or ER‐stress inducer, thapsigargin could partially stimulate or even repress some of them in HCC cells. Moreover, six of these identified genes contain potential ER stress‐responsive elements and/or unfolded protein response elements in their 5′ regulatory regions.

Down-regulation of c-myc and bcl-2 gene expression in PU.1-induced apoptosis in murine erythroleukemia cells

We found that over‐expression of PU.1, a member of the ets family of transcription factors, induces apoptotic cell death along with differentiation of DMSO stimulation in murine erythroleukemia (MEL) cells. To elucidate the molecular mechanisms of apoptosis, cell‐cycle distribution and expression of several genes encoding apoptosis‐promoting and ‐inhibiting factors were analyzed during the process of PU.1‐induced apoptosis. FACS analysis revealed that cells were accumulated in the G0/G1 phase of the cell cycle before apoptosis. Morphological analysis of PI‐stained nuclei of the apoptotic cells sorted by a FACScan showed 22.6% in G0/G1, 35.8% in S and 8.5% in G2/M phase by fluorescent microscopy after cell sorting, suggesting that PU.1‐induced apoptosis in MEL cells occurs in G0/G1 through S phases. Semi‐quantitative RT‐PCR revealed that expression of c‐myc and bcl‐2 genes was reduced during the apoptotic process, while expression of bax and bcl‐XL genes was not changed. Expression of the p53 gene was reduced rather than enhanced, suggesting that PU.1‐induced apoptosis in MEL cells is p53‐independent. Apoptosis was inhibited by adding 30% serum in culture, while no reduction of c‐myc and bcl‐2 gene expression was observed. Forced expression of the c‐myc, bcl‐2 and bcl‐XL genes protected MEL cells from apoptosis. Our results suggest that a reduction of at least 2 important apoptosis‐inhibiting factors, c‐Myc and Bcl‐2, is involved in PU.1‐induced apoptosis in MEL cells. Int. J. Cancer 76:523–530, 1998.© 1998 Wiley‐Liss, Inc.

Enhanced expression of mRNAs of antisecretory factor-1, gp96, DAD1 and CDC34 in human hepatocellular carcinomas

To identify differentially expressed genes in hepatocarcinogenesis, we performed differential display analysis using surgically resected hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues. We identified four cDNA fragments upregulated in HCC samples, encoding antisecretory factor-1 (AF), gp96, DAD1 and CDC34. Northern blot analysis demonstrated that these mRNAs were expressed preferentially in HCCs compared with adjacent non-tumorous liver tissues or normal liver tissues from non-HCC patients. The expression of these mRNAs was increased along with the histological grading of HCC tissues. These mRNA levels were also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. We also demonstrate that sodium butyrate, an inducer of differentiation, downregulated the expression of AF and gp96 mRNAs, supporting in part our pathological observation. Immunohistochemical analysis revealed that gp96 and CDC34 proteins were preferentially accumulated in cytoplasm and nuclei of HCC cells, respectively. Overexpression of these genes could be an important manifestation of HCC phenotypes and should provide clues to understand the molecular basis of hepatocellular carcinogenesis.

The S29 ribosomal protein increases tumor suppressor activity of K rev-1 gene on v-K ras-transformed NIH3T3 cells☆

The human S29 ribosomal protein (S29 rp) cDNA has been isolated from differential hybridization screening of a colon carcinoma cDNA library. Northern blot analysis showed that the level of S29 rp mRNA was higher in undifferentiated HT29 human colon carcinoma cells than in a morphologically differentiated subclone under the same growth condition. Furthermore, the level of S29 rp mRNA was downregulated in rapidly proliferating HT29 cells, as compared to the contact inhibited cells. Interestingly, the amount of Krev-1 mRNA was inversely correlated with respect to the amount of S29 rp mRNA in these cells. To examine a functional link between S29 rp and Krev-1 protein, we co-transfected the expression vectors containing wild-type or mutant S29 rp and mutationally activated Krev-1(63E) cDNAs into the v-Ki-ras-transformed NIH3T3 (DT) cells, and observed the induction of flat revertants. Krev-1(63E) induced a certain amount of flat colonies, while S29 rp alone also induced flat colonies at low frequencies. Interestingly, revertant-inducing activity of Krev-1(63E) was significantly enhanced by S29 rp. We have also demonstrated that a zinc finger-like domain of S29 rp indeed has a zinc binding activity and a derivative, S29 rp(ms), which was unable to bind zinc ion but still retained revertant inducing activity by itself, could not functionally interact with Krev-1(63E) protein.

The knockdown of endogenous replication factor C4 decreases the growth and enhances the chemosensitivity of hepatocellular carcinoma cells

Aims: To identify differentially expressed genes and thereby detect potential molecular targets for future therapies directed against hepatocellular carcinoma (HCC).