Naohisa Yoshioka

Regulation of Late G1/S Phase Transition and APCCdh1 by Reactive Oxygen Species

ABSTRACT Proliferating cells have a higher metabolic rate than quiescent cells. To investigate the role of metabolism in cell cycle progression, we examined cell size, mitochondrial mass, and reactive oxygen species (ROS) levels in highly synchronized cell populations progressing from early G1 to S phase. We found that ROS steadily increased, compared to cell size and mitochondrial mass, through the cell cycle. Since ROS has been shown to influence cell proliferation and transformation, we hypothesized that ROS could contribute to cell cycle progression. Antioxidant treatment of cells induced a late-G1-phase cell cycle arrest characterized by continued cellular growth, active cyclin D-Cdk4/6 and active cyclin E-Cdk2 kinases, and inactive hyperphosphorylated pRb. However, antioxidant-treated cells failed to accumulate cyclin A protein, a requisite step for initiation of DNA synthesis. Further examination revealed that cyclin A continued to be ubiquitinated by the anaphase promoting complex (APC) and to be degraded by the proteasome. This antioxidant arrest could be rescued by overexpression of Emi1, an APC inhibitor. These observations reveal an intrinsic late-G1-phase checkpoint, after transition across the growth factor-dependent G1 restriction point, that links increased steady-state levels of endogenous ROS and cell cycle progression through continued activity of APC in association with Cdh1.

Link of a new type of apoptosis-inducing gene ASY/Nogo-B to human cancer.

Although apoptosis plays an essential role in the embryogenesis and homeostasis of multicellular organisms, this mechanism has not yet been fully clarified. We isolated a novel human apoptosis-inducing gene, ASY, which encodes an endoplasmic reticulum-targeting protein without any known apoptosis-related motifs. This gene is identical to the Nogo-B, a splice variant of the Nogo-A which has recently been shown to be an inhibitor of neuronal regeneration in the central nervous system. Ectopic expression of the ASY gene led to extensive apoptosis, particularly in cancer cells. Furthermore, transcription of the ASY gene was suppressed in small cell lung cancer. These results suggest that a new type of apoptosis-inducing gene, namely, ASY, may be involved in the development of certain types of cancer.

Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications

RNA interference (RNAi) has great potential to treat human disease. However, in vivo delivery of short interfering RNAs (siRNAs), which are negatively charged double-stranded RNA macromolecules, remains a major hurdle. Current siRNA delivery has begun to move away from large lipid and synthetic nanoparticles to more defined molecular conjugates. Here we address this issue by synthesis of short interfering ribonucleic neutrals (siRNNs) whose phosphate backbone contains neutral phosphotriester groups, allowing for delivery into cells. Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs, which induce robust RNAi responses. siRNNs have favorable drug-like properties, including high synthetic yields, serum stability and absence of innate immune responses. Unlike siRNAs, siRNNs avidly bind serum albumin to positively influence pharmacokinetic properties. Systemic delivery of siRNNs conjugated to a hepatocyte-specific targeting domain induced extended dose-dependent in vivo RNAi responses in mice. We believe that siRNNs represent a technology that will open new avenues for development of RNAi therapeutics.

Periostin is down-regulated in high grade human bladder cancers and suppresses in vitro cell invasiveness and in vivo metastasis of cancer cells

We have previously reported that expression of periostin mRNA is markedly reduced in a variety of human cancer cell lines, suggesting that downregulation of periostin mRNA expression is correlated with the development of human cancers. In our study, to clarify the role of the periostin in human bladder carcinogenesis, we examined the expression of periostin mRNA in normal bladder tissues, bladder cancer tissues and bladder cancer cell lines by Northern blot analysis and RT‐PCR analysis. Although the expression of periostin mRNA was detected in 100% (5/5) of normal bladder tissues, it was not detected in 3 human bladder cancer cell lines examined. It was also detected in 81.8% (9/11) of grade 1, 40.0% (4/10) of grade 2 and 33.3% (4/12) of grade 3 bladder cancer tissues, indicating that downregulation of periostin mRNA is significantly related to higher grade bladder cancer (p<0.05). To assess the tumor suppressor function of periostin, we investigated the ability of periostin gene to suppress malignant phenotypes of a bladder cancer cell line, SBT31A. Ectopic expression of periostin gene by a retrovirus vector suppressed in vitro cell invasiveness of the bladder cancer cells without affecting cell proliferation and tumor growth in nude mice. Periostin also suppressed in vivo lung metastasis of the mouse melanoma cell line, B16–F10. Mutational analysis revealed that the C‐terminal region of periostin was sufficient to suppress cell invasiveness and metastasis of the cancer cells. Periostin may play a role as a suppressor of invasion and metastasis in the progression of human bladder cancers.

A systems biology dynamical model of mammalian G1 cell cycle progression.

The current dogma of G1 cell‐cycle progression relies on growth factor‐induced increase of cyclin D:Cdk4/6 complex activity to partially inactivate pRb by phosphorylation and to sequester p27Kip1‐triggering activation of cyclin E:Cdk2 complexes that further inactivate pRb. pRb oscillates between an active, hypophosphorylated form associated with E2F transcription factors in early G1 phase and an inactive, hyperphosphorylated form in late G1, S and G2/M phases. However, under constant growth factor stimulation, cells show constitutively active cyclin D:Cdk4/6 throughout the cell cycle and thereby exclude cyclin D:Cdk4/6 inactivation of pRb. To address this paradox, we developed a mathematical model of G1 progression using physiological expression and activity profiles from synchronized cells exposed to constant growth factors and included a metabolically responsive, activating modifier of cyclin E:Cdk2. Our mathematical model accurately simulates G1 progression, recapitulates observations from targeted gene deletion studies and serves as a foundation for development of therapeutics targeting G1 cell‐cycle progression.

Isolation of Transformation Suppressor Genes by cDNA Subtraction: Lumican Suppresses Transformation Induced by v-src and v-K-ras

ABSTRACT We have reported that suppressive factors for transformation by viral oncogenes are expressed in primary rat embryo fibroblasts (REFs). To identify such transformation suppressor genes, we prepared a subtracted cDNA library by using REFs and a rat normal fibroblast cell line, F2408, and isolated 30 different cDNA clones whose mRNA expression was markedly reduced in F2408 cells relative to that in REFs. We referred to these as TRIF (transcript reduced in F2408) clones. Among these genes, we initially tested the suppressor activity for transformation on three TRIF genes, TRIF1 (neuronatin), TRIF2 (heparin-binding growth-associated molecule), and TRIF3 (lumican) by focus formation assay and found that lumican inhibited focus formation induced by activated H-ras in F2408 cells. Colony formation in soft agar induced by v-K-ras or v-src was also suppressed in F2408 clones stably expressing exogenous lumican without disturbing cell proliferation. Tumorigenicity in nude mice induced by these oncogenes was also suppressed in these lumican-expressing clones. These results indicate that lumican has the ability to suppress transformation by v-src and v-K-ras.

Pro‐apoptotic ASY/Nogo‐B protein associates with ASYIP

We have previously shown that ectopic expression of the ASY/Nogo‐B gene induced apoptosis in various cancer cell lines. Nogo‐A, a splice variant of the ASY, has been reported to have an inhibitory effect on neuronal regeneration in the central nervous system. To investigate the mechanism of ASY‐induced apoptosis or inhibition of neuronal regeneration, we cloned a cDNA for the ASY‐interacting protein from the human cDNA library using the yeast two‐hybrid method, and obtained a cDNA we designated as ASYIP. The ASYIP protein contains two hydrophobic regions and a double lysine endoplasmic reticulum (ER) retrieval motif at its C‐terminus, which was shown to be identical to RTN3, a reticulon family protein of unknown function. We showed that ASY and ASYIP proteins formed a complex also in human cells. Mutational analysis indicated that both of the hydrophobic regions of the ASYIP protein were required for the association. By immunofluorescence analysis, the ASYIP protein was shown to be co‐localized with ASY in the ER. Characterization of the ASYIP gene may be very useful in clarifying the mechanism of ASY‐induced apoptosis or Nogo‐involved inhibition of neuronal regeneration in the central nervous system. J. Cell. Physiol. 196: 312–318, 2003.

Reduction of syndecan-1 mRNA in cervical-carcinoma cells is involved with the 3' untranslated region.

Syndecan‐1 is a transmembrane proteoglycan expressed predominantly in epithelial cells. Studies with immunohistochemistry have shown that syndecan‐1 expression is reduced in carcinoma derived from human epidermis. Here we show that syndecan‐1 mRNA, which is abundant in human primary keratinocyte (HK) and HaCaT spontaneous immortalized keratinocyte, is decreased in cervical‐carcinoma cell lines. Further, in relation to a long and well‐conserved 3′ untranslated region (3′ UTR) of syndecan‐1 cDNA, we examined whether 3′ UTR is involved with syndecan‐1‐mRNA reduction in cervical‐carcinoma cells. A stable transfection experiment showed that addition of the 3′ UTR does not affect expression in HaCaT, but that syndecan‐1 cDNA containing the 3′ UTR is not expressed efficiently selectively in cervical‐carcinoma cell lines. The transient assay with CAT reporter plasmids linking the 3′ UTR confirmed this, and indicated that the 3′ end of the 3′ UTR (nt 2285–2410) is required to influence expression in cervical‐carcinoma cells. Further excessive expression of syndecan‐1 suppressed growth in cervical‐carcinoma cells. These results demonstrate that the reduction of syndecan‐1 mRNA involved with the 3′ untranslated region gives growth advantage to cervical‐carcinoma cells. Int. J. Cancer 80:527–532, 1999.

Isolation and characterization of the TIGA genes, whose transcripts are induced by growth arrest

We report here the isolation of 44 genes that are upregulated after serum starvation and/or contact inhibition. These genes have been termed TIGA, after Transcript Induced by Growth Arrest. We found that there are two kinds of G0 phases caused by serum starvation, namely, the shallow G0 (or G0/G1) and the deep G0 phases. The shallow G0 is induced by only a few hours of serum starvation, while deep G0 is generated after 3 days of serum starvation. We propose that mammalian cells enter deep G0 through a G0 gate, which is only opened on the third day of serum starvation. TIGA1, one of the uncharacterized TIGA genes, encodes a homolog of cyanate permease of bacteria and localizes in mitochondria. This suggests that Tiga1 is involved in the inorganic ion transport and metabolism needed to maintain the deep G0 phase. Ectopic expression of TIGA1 inhibited not only tumor cell proliferation but also anchorage-independent growth of cancer cell lines. A microsatellite marker, ENDL-1, allowed us to detect loss of heterozygosity around the TIGA1 gene region (5q21–22). Further analysis of the TIGA genes we have identified here may help us to better understand the mechanisms that regulate the G0 phase.

Malignant transformation of human diploid fibroblasts and suppression of their anchorage independence by introduction of chromosome 13.

Isolation of cell lines that display various degrees of transformed phenotypes may be very useful to clarify multistep mechanisms of oncogenesis, but malignant transformation of human diploid fibroblasts in culture is a very rare event. We attempted to isolate variously transformed cell lines from human diploid fibroblasts (RB) of a patient with hereditary retinoblastoma. The RB cells exhibited normal karyotypes with the exception of one copy of chromosome 13, which contained a large deletion at the q14–22 region, where the RB1 gene is located. By transfection with SV40 early genes and repeated passage, we succeeded in obtaining SV40‐transfected mortal, immortalized, anchorage‐independent, and tumorigenic RB cell lines. DNA fingerprinting showed that these cell lines were not contaminants, but derivatives of the original RB cells. The remaining RB1 allele may be wild‐type even in the malignant cell lines, because the expression and the LT‐binding ability were normal. Furthermore, we did not find any homozygous loss in 16 polymorphic markers located in the 13q14–22 region in the transformed cell lines. However, introduction of a copy of a normal chromosome 13 into the anchorage‐independent cell line suppressed its anchorage‐independent growth ability. All these data, together with the fact that the RB cells containing the deletion progressed to a tumorigenic state spontaneously, but normal fibroblasts did not, raise the possibility that a new tumor suppressor gene, located at 13q14–22, may play a critical role in neoplastic transformation. We conclude that these RB cell lines provide an excellent system for identification of genes involved in malignant transformation of human cells. Genes Chromosomes Cancer 26:47–53, 1999.