Hiroki Sawa

Alternatively spliced forms of cyclin D1 modulate entry into the cell cycle in an inverse manner.

Alternative splicing of cyclin D1 gene mRNA has recently been demonstrated. The novel transcript shows no splicing at the downstream exon 4 boundary and encodes a protein with an altered carboxyl-terminal domain that is a cyclin D1 variant; exon 5 is not included in the coding sequence which terminates downstream of exon 4. We here produced cells that exogenously express each form of cyclin D1 and analysed their cell cycle regulation. We found that (1) alternative splicing forms of cyclin D1 modulated entry into the cell cycle in an inverse manner; (2) both splicing forms suppressed cell growth; and (3) cells overexpressing form [a] were inhibited from entry into and completion of the S phase, although form [b]-expressing cells showed no reduction of G1- to S transition. We also found that overexpression of either cyclin D1 form up-regulated Rb gene products, suggesting that this up-regulation may be one of the causes of growth suppression in cyclin D1 overexpressing cells.

Expression and functional role of syntaxin 1/HPC-1 in pancreatic beta cells. Syntaxin 1A, but not 1B, plays a negative role in regulatory insulin release pathway.

Syntaxin 1/HPC-1 is an integral membrane protein, which is thought to be implicated in the regulation of synaptic neurotransmitter release. We investigated syntaxin 1 expression in pancreatic β cells and the functional role of syntaxin 1 in the insulin release mechanism. Expression of syntaxin 1A, but not 1B, was detected in mouse isolated islets by the reverse transcriptase-polymerase chain reaction procedure. An immunoprecipitation study of metabolically labeled islets with an anti-rat syntaxin 1/HPC-1 antibody demonstrated syntaxin 1A protein with an apparent molecular mass of ∼35 kDa. Immunohistochemistry of the mouse pancreas demonstrated that syntaxin 1/HPC-1 was present in the plasma membranes of the islets of Langerhans. In order to determine the functional role of syntaxin 1 in pancreatic β-cells, rat syntaxin 1A or 1B was overexpressed in mouse βTC3 cells using the transient transfection procedure. Transfection of βTC3 cells with either syntaxin 1 resulted in approximately 7-fold increases in their immunodetectable protein levels. Glucose-stimulated insulin release by syntaxin 1A-overexpressing cells was suppressed to about 50% of the level in control cells, whereas insulin release by syntaxin 1B-overexpressing and control cells did not differ. Next, we established stable βTC3 cell lines that overexpressed syntaxin 1A and used them to evaluate the effect of syntaxin 1A on the regulatory insulin release pathway. Two insulin secretogogues, 4-β-phorbol 12-myristate 13-acetate or forskolin, increased insulin release by untransfected βTC3 cells markedly, but their effects were diminished in syntaxin 1A-overexpressing βTC3 cells. Glucose-unstimulated insulin release and the proinsulin biosynthetic rate were not affected by syntaxin 1A overexpression, indicating a specific role of syntaxin 1A in the regulatory insulin release pathway. Finally, in vitro binding assays showed that syntaxin 1A binds to insulin secretory granules, indicating an inhibitory role of syntaxin 1A in insulin exocytosis via its interaction with vesicular proteins. These results demonstrate that syntaxin 1A is expressed in the islets of Langerhans and functions as a negative regulator in the regulatory insulin release pathway.

Neuron-specific glucose transporter (NSGT): CNS distribution of GLUT3 rat glucose transporter (RGT3) in rat central neurons.

The identity of the glucose transporters (GLUT) expressed in neurons in situ has yet to be fully established. In the present study we have isolated the GLUT3 (RGT3) cDNA and produced anti RGT3 polyclonal antibody allowing us to investigate the cellular localization and tissue distributions of RGT3 mRNA and protein in the central nervous system of the rat by the methods of in situ hybridization and immunohistochemistry. Here we demonstrate the direct evidence that RGT3 is present in neurons in adult rat brain. In situ hybridization showed the expression of RGT3 mRNA mostly in the regions of hippocampus, cerebral cortex, striatum, and the granule cell layer of the cerebellum, indicating that RGT3 mRNA is predominantly expressed within neurons. Immunohistochemistry showed that RGT3 protein is widely distributed in the rat brain, and concentrated on the plasma membrane of neurons. Double labeling studies with anti‐RGT3, glial fibrillary acidic protein (GFAP), and neuron specific enolase (NSE) antibodies revealed the specific expression of RGT3 protein in neurons. Thus, RGT3 is indicated to be a neuron specific glucose transporter isoform (NSGT), and suggested to play a functionally significant role in rat central neurons.

Histone deacetylase inhibitors such as sodium butyrate and trichostatin A induce apoptosis through an increase of the bcl-2-related protein Bad.

The effects of sodium butyrate (SB) and trichostatin A (TSA) on cell proliferation and apoptosis against human glioma T98G, U251MG, and U87MG cells were investigated. Upon exposure to either SB or TSA, cell proliferation was reduced, and apoptosis detected by DNA fragmentation analysis and the cleavage of CPP32 was induced. Previously, we reported that SB increased the expression levels of p21 (WAF-1) and inhibited G1-S transition of the cell cycle. In this study, we showed that TSA also increased p21 expression, suggesting that histone deacetylase (HDAC) inhibitors may up-regulate p21 protein in common and thus arrest proliferation in the G1 phase of the cell cycle. To further determine the underlying molecular mechanisms of apoptosis with either SB or TSA treatment, we studied the expression levels of apoptosisrelated proteins in human glioma cells. SB increased the expression of the Bad protein, although the expression of Bcl-2, Bcl-xL, Bax, and Fas was not changed by the addition of SB. TSA treatment also up-regulated the expression of Bad protein. The results suggest that HDAC inhibitors such as SB and TSA induce apoptosis through an increase in Bad protein in human glioma cells in vitro.

Expression of Facilitative Glucose Transporter Isoforms in Human Brain Tumors

The expression of facilitative glucose transporter (GLUT) isoforms in human astrocytic tumors was examined. Reverse transcriptase‐polymerase chain reaction of a surgically biopsied glioblastoma was carried out using the degenerative oligonucleotide primers corresponding to the sequences of the human facilitative glucose transporter family, and polymerase chain reaction products were hybridized with human GLUT1, GLUT2, GLUT3, GLUT4, and GLUT5 cDNA probes. The results showed that a biopsied glioblastoma expressed GLUT1, GLUT3, and GLUT4 glucose transporter genes. Northern blot analysis of total RNA (10 μg) from a biopsied glioblastoma showed the transcripts of only GLUT1 and GLUT3, suggesting that the expression of insulin‐responsive glucose transporter GLUT4 mRNA is relatively low. Immunoblot analysis of biopsied glioblastoma tissues by polyclonal antibodies against the C‐terminal synthetic peptides of GLUT1, GLUT3, and GLUT4 showed a single band of each polypeptide. However, elevated expression of GLUT1 and GLUT3 glucose transporters was not observed in the glioblastoma. Astrocytic tumor tissues (n = 14) were also examined immunohistochemically. Reactive products for GLUT1 were observed in the luminal surface of capillaries in all cases, whereas tumor cells were positive for GLUT1 in only two of 14 cases. GLUT3 was positive in astrocytic tumor cells in all cases. Three of 14 cases expressed the GLUT4 protein, which was localized in the cytoplasm of tumor cells. These results suggest that the facilitative glucose transport may be altered in astrocytic tumor cells and thus display a significant change in glucose metabolism.

Over-expression of cyclin D1 induces glioma invasion by increasing matrix metalloproteinase activity and cell motility.

In order to define the role of cyclin D1 in the progression of malignant glioma, cells over‐expressing cyclin D1 were constructed (a‐1 cells). They exhibited significantly increased invasiveness as compared with mock‐transfected cells. Since cellular invasion is thought to depend on extracellular‐matrix degradation, we determined whether cyclin‐D1 expression modifies the activity of matrix metalloproteinases (MMPs). Increased gelatinolytic activity of latent type MMP‐2 (proMMP‐2) and active MMP‐2 was observed in a‐1 cells. Moreover, cyclin‐D1 expression was associated with increased activation of proMMP‐9 through MMP‐3. Wound assays showed an increase of cell motility in a‐1 cells. Cyclin‐D1 expression was found to be associated with up‐regulation of Rac1, which modulates the formation of ruffling membranes and cell motility. Our results show that cyclin D1 may modulate invasive ability by increasing MMP activity and cell motility, and suggests a novel function of cyclin D1 in the progression of malignant gliomas. Int. J. Cancer 83:387–392, 1999.

Histone deacetylase inhibitor, FK228, induces apoptosis and suppresses cell proliferation of human glioblastoma cells in vitro and in vivo

We investigated the effects of FK228 on cell proliferation and apoptosis against human glioblastoma (GM) T98G, U251MG, and U87MG cells. Upon exposure to FK228, cell proliferation was inhibited, and apoptosis detected by the cleavage of CPP32 was induced. FK228 increased the expression levels of p21 (WAF-1) and of pro-apoptotic Bad protein in all GM cells. Furthermore, FK228 treatment also reduced the anti-apoptotic protein Bcl-xL in all GM cells and anti-apoptotic Bcl-2 in U87MG cells, thereby shifting the cellular equilibrium from life to death. An increased accumulation of histone H4 was detected in the p21 (WAF-1) promoter and the structural gene (exon 2) and the Bad structural gene (exon 2 and 3) upon treatment with FK228, as assessed by chromatin immunoprecipitation (ChIP) assay. Thus, the results indicated that an increased expression of p21 (WAF1) and Bad due to FK228 is regulated, at least in part, by the degree of acetylation of the gene-associated histone. We also found that FK228 inhibits cellular invasiveness and decreases MMP-2 activity. In addition, the growth of transplanted human GM m-3 cells into the subcutaneous tissue of hereditary athymic mice was significantly inhibited, and apoptosis was induced with FK228 treatment. The results suggested that FK228 might be useful in the treatment of human GM, although further studies will be needed.

Expression of glucose transporters in rat brain following transient focal ischemic injury.

Abstract: We have investigated the serial changes in the transcription and translation of the rat glucose transporter (GLUT) 1 and 3 genes after 3 h of middle cerebral artery (MCA) occlusion followed by reperfusion. Northern blot analysis and in situ hybridization study were performed to determine the chronological change and regional expression. In the ipsilateral anterior cerebral artery (ACA) cortex, GLUT1 mRNA expression was increased at 12 h (11.6‐fold) of reperfusion, and its expression was detected not only in vascular endothelial cells but also in neurons. At 48 h of reperfusion, GLUT3 mRNA expression was increased in the ipsilateral ACA (8.6‐fold) and in the contralateral MCA cortex (9.1‐fold). Immunohistochemical study failed to show GLUT1 protein synthesis in neurons in the ipsilateral ACA cortex. The immunoreactivity of GLUT3 protein was increased in neurons in ipsilateral ACA cortex and contralateral MCA cortex. Our results suggest that the expression of GLUT1 and GLUT3 is controlled differently after transient focal ischemic conditions. Furthermore, the postischemic localizations of both GLUT1 and GLUT3 expressions may be altered from the normal physiological expression pattern, which may be of importance in investigating postischemic cell function.

Histone deacetylase inhibitors such as sodium butyrate and trichostatin A inhibit vascular endothelial growth factor (VEGF) secretion from human glioblastoma cells

We investigated the effects of histone deacetylase (HDAC) inhibitors such as sodium butyrate (SB) and trichostatin A (TSA) on the expression of vascular endothelial growth factor (VEGF) by human glioblastoma T98G, U251MG, and U87MG cells. The glioblastoma cells secreted three VEGF isoforms, VEGF (189), (165), and (121), although the expression levels of VEGF differed between the cell types. Treatment with either 5mM SB or 100ng/ml TSA reduced VEGF secretion in conditioned media and reduced VEGF mRNA expression. We also studied the expression of VEGF-B,-C, and-D mRNA in human glioblastoma cells and their modulation by HDAC inhibitors. The PCR products of VEGF-B (357 bp), VEGF-C (501 bp), and VEGF-D (484 bp) were amplified in all glioblastoma cells examined. Treatment with SB reduced the expression of VEGF-D mRNA in U251MG cells and the expression of VEGF-B mRNA in U87MG cells. TSA treatment reduced the expression of VEGF-D in U251MG cells. These results suggest that HDAC inhibitors reduce VEGF secretion and modulate the expression of the other VEGF family members, and therefore may inhibit angiogenesis in glioblastoma tissues.

Expression of p53, MDM2 protein and Ki-67 antigen in recurrent meningiomas.

Association of p53 gene abnormalities with tumor progression and prognosis of many neoplasms has been demonstrated, but little is known about the clinical significance of p53 abnormalities in meningiomas. The significance of p53 protein expression in recurrent meningiomas and its relationships with MDM2 protein and proliferation activity were investigated by analyzing 39 meningiomas immunohistochemically. p53 protein was expressed in 11 (35%) of 31 non-recurrent and 7 (88%) of 8 recurrent meningiomas. A high frequency of p53 expression was observed in recurrent meningiomas, which tended to have a high p53 positive index (p53 PI), indicating that p53 immunoreactivity may be a marker for predicting tumor recurrence. Four recurrent meningiomas with high p53 PIs were analyzed by the polymerase chain reaction-single strand conformation polymorphism method to detect p53 gene mutations, but none were found in exons 4–8 of this gene. Fifteen (71%) of 21 MDM2-positive and 3 (17%) of 18 MDM2-negative tumors expressed p53 protein, showing that MDM2 expression was more common in meningiomas with p53 expression. p53 immunoreactivity in the absence of mutation may indicate stabilization of the wild type through interaction with the MDM2 protein. The Ki-67/MIB-1 proliferation index (MIB-1 PI) correlated well with recurrence. The p53-positive tumors had a significantly higher mean MIB-1 PI than p53-negative tumors, suggesting that wild-type p53 inactivation by the MDM2 protein may be involved in controlling the proliferative activity in meningiomas. In conclusion, immunohistochemical examination for p53 protein as well as proliferative activity may help predict the malignant potential of tumor recurrence.