Biomedical subjects

E Hara

Publications and source records attributed to E Hara.

At least 19 recordsLinked to original sources

PubMed1999

Induced expression of p16(INK4a) inhibits both CDK4- and CDK2-associated kinase activity by reassortment of cyclin-CDK-inhibitor complexes.

To investigate the mode of action of the p16(INK4a) tumor suppressor protein, we have established U2-OS cells in which the expression of p16(INK4a) can be regulated by addition or removal of isopropyl-beta-D-thiogalactopyranoside. As expected, induction of p16(INK4a) results in a G1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16(INK4a) also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27(KIP1). Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16(INK4a), this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16(INK4a). Sequestration of CDK4 by p16(INK4a) allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16(INK4a), p27(KIP1) appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27(KIP1) and p21(CIP1). Significantly, p16(INK4a) itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.

CDC2-CDC28 Kinases↗

PubMed1998

Differential activity of a variant form of the human Id-1 protein generated by alternative splicing.

Members of the Id family of helix-loop-helix proteins are ubiquitously expressed and dimerize with members of the class A and class B basic helix-loop-helix proteins. Due to the absence of a basic region, Id proteins act as dominant-negative antagonists of basic helix-loop-helix transcription factors, which regulate cell growth and differentiation in diverse cell types. Recent findings suggest that the functions of Id proteins are well regulated at both the transcriptional level and the post-transcriptional level. We show here that the alternative splicing variant of human Id-1 protein possesses a different binding specificity for basic helix-loop-helix transcription factors and is expressed in a cell cycle-dependent manner. Therefore, alternative splicing of Id-1 could provide a post-transcriptional mechanism to regulate Id-1 function.

Alternative Splicing↗

PubMed1998

The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2.

The two distinct proteins encoded by the CDKN2A locus are specified by translating the common second exon in alternative reading frames. The product of the alpha transcript, p16(INK4a), is a recognized tumour suppressor that induces a G1 cell cycle arrest by inhibiting the phosphorylation of the retinoblastoma protein by the cyclin-dependent kinases, CDK4 and CDK6. In contrast, the product of the human CDKN2A beta transcript, p14(ARF), activates a p53 response manifest in elevated levels of MDM2 and p21(CIP1) and cell cycle arrest in both G1 and G2/M. As a consequence, p14(ARF)-induced cell cycle arrest is p53 dependent and can be abrogated by the co-expression of human papilloma virus E6 protein. p14(ARF) acts by binding directly to MDM2, resulting in the stabilization of both p53 and MDM2. Conversely, p53 negatively regulates p14(ARF) expression and there is an inverse correlation between p14(ARF) expression and p53 function in human tumour cell lines. However, p14(ARF) expression is not involved in the response to DNA damage. These results place p14(ARF) in an independent pathway upstream of p53 and imply that CDKN2A encodes two proteins that are involved in tumour suppression.

Alternative Splicing↗

PubMed1998

Features of replicative senescence induced by direct addition of antennapedia-p16INK4A fusion protein to human diploid fibroblasts.

The p16INK4A cyclin-dependent kinase (Cdk) inhibitor is now recognized as a major tumor suppressor that is inactivated by a variety of mechanisms in a wide range of human cancers. It is also implicated in the mechanisms underlying replicative senescence since p16INK4A RNA and protein accumulate as cells approach their proscribed limit of population doublings in tissue culture. To obtain further evidence of its role in senescence, we have sought ways of overexpressing p16INK4A in primary human diploid fibroblasts (HDF). To circumvent the low transfection efficiency of primary cells we have exploited a recombinant form of the full-length p16INK4A protein fused to a 16 amino acid peptide from the Drosophila antennapedia protein. This peptide has the capacity to cross both cytoplasmic and nuclear membranes allowing the direct introduction of the active protein to primary cells. Here, we show that antennapedia-tagged wild-type p16INK4A protein, but not a functionally compromised tumor-specific variant, causes G1 arrest in early passage HDFs by inhibiting the phosphorylation of the retinoblastoma protein. Significantly, the arrested cells display several phenotypic features that are considered characteristic of senescent cells. These data support a role for p16INK4A in replicative senescence and raise the possibility of using the antennapedia-tagged protein therapeutically.

Antennapedia Homeodomain Protein↗

PubMed1998

Production and secretion of endothelin-1 by cultured choroid plexus carcinoma cells.

Choroid plexus carcinoma is a rare neoplasm derived from the epithelium of the choroid plexus. The production and secretion of endothelin-1 (ET-1) by cultured human choroid plexus carcinoma cells were studied by radioimmunoassay and Northern blot analysis. Immunoreactive (IR)-ET was detected in the culture medium (2.78 +/- 0.12 fmol/10(5) cells/24 h; n = 5; mean +/- SEM) but not in the unconditioned medium. Reverse-phase high-performance liquid chromatography of the extract of the culture medium showed a single peak eluting in the position of ET-1. Treatment with tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) or a combination of interferon-gamma (IFN-gamma), TNF-alpha, and IL-1 beta caused significant increases in the IR-ET levels in the culture medium. Northern blot analysis of total RNA showed the expression of ET-1 mRNA in choroid plexus carcinoma cells. The expression levels of ET-1 mRNA were increased by treatment with a combination of IFN-gamma, TNF-alpha, and IL-1 beta. The present study has shown the production and secretion of ET-1 by cultured human choroid plexus carcinoma cells and suggests the possibility that ET-1 formation is related to the pathophysiology of this tumor.

Blotting, Northern↗

PubMed1998

Production and secretion of two vasoactive peptides, endothelin-1 and adrenomedullin, by a colorectal adenocarcinoma cell line, DLD-1.

Production and secretion of endothelin-1 (ET-1) and adrenomedullin (ADM) by a cultured human colorectal adenocarcinoma cell line, DLD-1, were studied by radioimmunoassay and Northern blot analysis. Both immunoreactive (IR)-ET and IR-ADM were detected by radioimmunoassay in the culture medium of DLD-1 (IR-ET 0.86 +/- 0.05 fmol/10(5) cells/ 24 h; IR-ADM 1.20 +/- 0.09 fmol/10(5) cells/24 h; n = 5, mean +/- SEM). An analysis by reverse-phase high-performance liquid chromatography (HPLC) of the IR-ET in the culture medium showed a major immunoreactive peak in the position of ET-1. Reverse-phase HPLC of the IR-ADM in the medium showed three immunoreactive peaks, one of which eluted in the position of human ADM. Northern blot analysis showed the expression of ET-1 mRNA and ADM mRNA in the DLD-1 cells. Treatment with interferon-gamma (1-100 U/ml) for 24 h decreased the IR-ET levels in the culture medium but significantly increased IR-ADM levels. This study has shown the production and secretion of two vasoactive peptides, ET-1 and ADM, by DLD-1 colorectal adenocarcinoma cells. The secretion of IR-ET was decreased by treatment with interferon-gamma. These findings suggest possible pathophysiologic roles for ET-1 and ADM in colon mucosal epithelial cells and tumors derived from them.

Adenocarcinoma↗

PubMed1998

[Augmentation of anti-Candida activity of neutrophils by macrophages treated with itraconazole].

Effects of itraconazole (ITCZ) on anti-Candida activity of a macrophage (MP)-neutrophil axis were examined in vitro. Murine peritoneal MP, stimulated by an enterococcal preparation FK-23, released tumor necrosis factor a (TNF) into the culture medium in 20 hr-culture period. This level of TNF production by the stimulated MP, which were measured by a bioassay or immunological assay, was heightened by co-stimulation with 0.5 mg/ml of ITCZ. Supernatants obtained from the culture medium of FK-23 and ITCZ-treated MP not only contained about 10 ng/ml of TNF but also had the capacity to enhance anti-Candida activity of murine neutrophils. This enhancement was blocked by pretreatment of the MP-supernatant with anti-TNF antibody. Observation by scanning electron microscope suggested that cell walls of the candida mycelia, the growth of which was inhibited by the neutrophils in the presence of either the MP-supernatant or TNF, sustained similar structural damage. These results suggest that ITCZ augments anti-Candida activity of neutrophils through enhanced production of TNF by MP in vitro.

Animals↗

PubMed1997

Accumulation of p16INK4a in mouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status.

Viral transformation of mouse and human fibroblasts has very different effects on the composition of cyclin-dependent kinase (Cdk) complexes. In human cells transformed by the large T-antigen of simian virus 40 (SV40 T-Ag) and human tumour cell lines that lack a functional retinoblastoma gene product (pRb) no cyclin D1-Cdk4 complexes can be detected because all the available Cdk4 is associated with the Cdk-inhibitor p16INK4a. In contrast, SV40-transformed mouse cells and fibroblasts from Rh1-nullizygous mouse embryos contain normal levels of cyclin D1-Cdk4 complexes. To investigate this species difference, we have compared the biochemical properties and expression of mouse p16INK4a with that of its human counterpart. There is a marked increase in p16 RNA and protein levels as primary embryo fibroblasts approach their finite lifespan in culture, but mouse p16 expression does not appear to be influenced by the status of pRb. Transformed or spontaneously immortalized mouse cells therefore do not achieve the very high levels of p16 characteristic of pRb-negative human cell lines. We suggest that these differences may be related to the different frequencies with which mouse and human cells can be immortalized in culture.

3T3 Cells↗

PubMed1997

Genomic organization, sequence, and chromosomal localization of the human helix-loop-helix Id1 gene.

The helix-loop-helix protein Id-1 regulates growth and differentiation in many mammalian cells. In human fibroblasts, Id1 and Id1', a putative splicing variant, are cell cycle regulated, essential for proliferation, repressed by senescence, and overexpressed by some tumor cells. To better understand Id1, we determined the complete sequence, transcriptional start, and localization of the human Id1 gene. Human Id1 has two exons (426 bp and 42 bp), separated by an intron (239 bp). Id1' results from failure to splice the intron, which encodes 7 amino acids prior to a stop codon. Thus, Id1 and Id1' proteins differ only at the extreme C-terminus. Id1 transcription initiated 96 bp upstream of the initiation AUG; 2 kb of upstream sequence stimulated transcription of a reporter gene. Human Id1 maps to chromosome 20 at q11, very close to the centromere but outside the amplicons frequently found in human cancers.

Amino Acid Sequence↗

PubMed1997

Cdk2-dependent phosphorylation of Id2 modulates activity of E2A-related transcription factors.

The helix-loop-helix (HLH) protein Id2 is thought to affect the balance between cell growth and differentiation by negatively regulating the function of basic-helix-loop-helix (bHLH) transcription factors. Id2 acts by forming heterodimers that are unable to bind to specific (E-box) DNA sequences. Here we show that this activity can be overcome by phosphorylation of a serine residue within a consensus target site for cyclin-dependent kinases (Cdks). In vitro, Id2 can be phosphorylated by either cyclin E-Cdk2 or cyclin A-Cdk2 but not by cyclin D-dependent kinases. Analogous phosphorylation occurs in serum-stimulated human diploid fibroblasts at a time in late G1 consistent with the appearance of active cyclin E-Cdk2. The phosphorylation of Id2 in these cells correlates with the restoration of a distinct E-box-dependent DNA-binding complex, suggesting that the levels of this complex are modulated by both the abundance and phosphorylation status of Id2. These data provide a link between cyclin-dependent kinases and bHLH transcription factors that may be critical for the regulation of cell proliferation and differentiation.

3T3 Cells↗

PubMed1997

Production and secretion of adrenomedullin from glial cell tumors and its effects on cAMP production.

The expression of adrenomedullin (ADM) and its mRNA was studied in human glial cell tumors and cultured glioblastoma cell lines, T98G and A172. Northern blot analysis showed that ADM mRNA was expressed in all brain tumors examined (three anaplastic astrocytomas and two glioblastomas multiforme) and in the glioblastoma cell lines. Immunoreactive (IR-) ADM was detectable in these brain tumors by radioimmunoassay (0.31-2.0 pmol/g wet weight), except for one anaplastic astrocytoma. Reverse phase high performance liquid chromatography of the tumor extracts showed a single peak eluting in the position of ADM-(1-52). IR-ADM concentrations in the cultured media of T98G cells were 205.5 +/- 8.4 fmol/10(5) cells/24 h (mean +/- SEM, n = 5). Treatment of T98G cells with interferon gamma or interleukin 1 beta increased the expression levels of ADM mRNA and the IR-ADM concentrations in the cultured media, whereas tumor necrosis factor alpha decreased them in a dose-dependent manner. Treatment with synthetic ADM-(1-52) (10(-8) or 10(-7) mol/l) increased the cAMP concentrations in the cultured media of T98G cells. These findings suggest that ADM is secreted from glial cell tumors and is related to the pathophysiology of these tumors.

Adrenomedullin↗

PubMed1997

Elschnig pearl formation along the posterior capsulotomy margin after neodymium:YAG capsulotomy.

PURPOSE: To determine the incidence of Elschnig pearl formation along the capsulotomy margin (string of pearls) after neodymium:YAG (Nd:YAG) laser posterior capsulotomy and to elucidate its clinical features, predisposing factors, effect on visual function, and association with additional capsulotomy. SETTING: Keio University Hospital, Tokyo, Japan. METHODS: The records of 418 eyes that had had Nd:YAG posterior capsulotomy after cataract surgery were retrospectively reviewed. Of those, 315 were excluded for short follow-up (fewer than 12 months) or insufficient clinical examination data. In the remaining 103 eyes, the incidence of string of pearls was calculated, and its clinical features, predisposing factors, effect on visual function, and correlation with additional capsulotomy were evaluated. RESULTS: String of pearls was identified in 49 eyes (47.6%); 37 (75.5%) developed pearls within 1 year after Nd:YAG capsulotomy. The incidence was significantly higher in patients having intraocular lens implantation and continuous curvilinear capsulorhexis (CCC) than in those without (95.9 versus 61.1% and 97.9 versus 55.6%, respectively). No significant differences were found in patient age and sex, total Nd:YAG energy, and the presence of diabetes mellitus or high myopia. String of pearls caused visual disturbances in 17 eyes (34.7%). The rate of repeat capsulotomy was higher in patients with string of pearls than in those without (36.7 and 9.3%, respectively). CONCLUSIONS: String of pearls formation was a common and significant complication after Nd:YAG posterior capsulotomy. Intraocular lens implantation and CCC may promote its formation.

Adult↗

PubMed1997

Production and secretion of adrenomedullin by cultured choroid plexus carcinoma cells.

Adrenomedullin is a potent vasodilator peptide that was originally isolated from pheochromocytoma. The production and secretion of adrenomedullin by cultured choroid plexus carcinoma cells were studied by radioimmunoassay and northern blot hybridization. Choroid plexus carcinoma is a rare malignant tumor derived from the epithelium of the choroid plexus. Immunoreactive adrenomedullin was detected in the conditioned medium of choroid plexus carcinoma cells (40.8 +/- 7.5 fmol/10(5) cells/24 h; mean +/- SEM, n = 5). Reverse-phase HPLC of the conditioned medium showed one major peak of the immunoreactive peptide eluting in the position of synthetic human adrenomedullin and two smaller peaks eluting earlier. Addition of interleukin-1 beta (10 ng/ml) alone or in combination with three cytokines, interferon-gamma (100 U/ml), tumor necrosis factor-alpha (20 ng/ml), and interleukin-1 beta (10 ng/ml), caused significant increases in the immunoreactive adrenomedullin concentrations in the medium (approximately 175 and 293% of the control level, respectively). Northern blot analysis showed the expression of 1.6-kb adrenomedullin mRNA in the total RNA sample prepared from cultured choroid plexus carcinoma cells. Treatment with either interleukin-1 beta or the combination of three cytokines caused significant increases in levels of adrenomedullin mRNA in parallel with those in immunoreactive adrenomedullin concentrations in the conditioned medium. These findings raise a possibility that adrenomedullin is secreted from the choroid plexus and has physiological roles in the CNS via the CSF. In addition, adrenomedullin secreted from choroid plexus carcinoma may be related to the pathophysiology of the tumor.

Adrenomedullin↗

PubMed1997

Possible implications of the induction of human heme oxygenase-1 by nitric oxide donors.

To explore the involvement of nitric oxide (NO) in the induction of heme oxygenase-1, an essential enzyme in heme catabolism, we studied the effects of NO donors on the expression of heme oxygenase-1 mRNA in HeLa human cervical cancer cells. Treatment with each of three NO donors, sodium nitroprusside, 3-morpholinosydnonimine, and S-nitroso-L-glutathione, caused noticeable increases in the expression levels of heme oxygenase- mRNA, but not heme oxygenase-2 mRNA. On the other hand, nitrite or 8-bromo cGMP exerted no noticeable effect on the levels of heme oxygenase-1 mRNA. We showed that sodium nitroprusside also increased the levels of heme oxygenase-1 protein. The sodium nitroprusside-mediated increase in heme oxygenase-1 mRNA levels was abolished by treatment with actinomycin D. The expression levels of heme oxygenase-1 mRNA were also increased by NO donors in human melanoma and neuroblastoma cell lines. Thus, the observed induction of heme oxygenase-1 may represent an important response to NO or NO-related oxidative stress. The half lives of heme oxygenase-1 and heme oxygenase-2 mRNAs were estimated to be about 3.2 h and more than 5 h, respectively.

Cell Lineage↗

PubMed1997

Regulation of Id3 cell cycle function by Cdk-2-dependent phosphorylation.

The functions of basic helix-loop-helix (bHLH) transcription factors in activating differentiation-linked gene expression and in inducing G1 cell cycle arrest are negatively regulated by members of the Id family of HLH proteins. These bHLH antagonists are induced during a mitogenic signalling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences. Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser5) in Id2 has been shown to occur during late G1-to-S phase transition of the cell cycle, and this neutralizes the function of Id2 in abrogating E-box-dependent bHLH homo- or heterodimer complex formation in vitro (E. Hara, M. Hall, and G. Peters, EMBO J. 16:332-342, 1997). We now show that an analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH homo- or heterodimer complex formation in vitro and E-box-dependent reporter gene function in vivo. Furthermore, compared with wild-type Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to promote cell cycle S phase entry in transfected fibroblasts, whereas an Id3 Ala5 mutant (ablating phosphorylation) displays an activity significantly greater than that of wild-type Id3 protein. Cdk2-dependent phosphorylation therefore provides a switch during late G1-to-S phase that both nullifies an early G1 cell cycle regulatory function of Id3 and modulates its target bHLH specificity. These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not simply a function of its ability to modulate bHLH heterodimer-dependent gene expression and establish a biologically important mechanism through which Cdk2 and Id-bHLH functions are integrated in the coordination of cell proliferation and differentiation.

Animals↗

PubMed1997

Regulation and function of the cyclin-dependent kinase inhibitor p16CDKN2.

Many human tumours show perturbations of a pathway that includes the D-cyclins, their associated cyclin-dependent kinases, and specific kinase inhibitors. The focal point of this pathway is the product of the retinoblastoma tumour suppressor gene, pRb, which imposes a block on G1 phase progression. Thus, the major role of the cyclin D-dependent kinases is to overcome this block by initiating the phosphorylation of pRb. Excessive activity of this pathway is likely to lead to excessive cell proliferation. Conversely, accumulation of the inhibitors is associated with the cessation of cell division.

Carrier Proteins↗

PubMed1996

Overexpression of cysteine sulfinic acid decarboxylase stimulated by hepatocarcinogenesis results in autoantibody production in rats.

We developed a novel and efficient cDNA subtraction method to isolate rat hepatocellular carcinoma (HCC)-related genes. cDNAs from Solt-Farber procedure-driven HCCs were synthesized on Latex beads. The subtraction was accomplished by a simple centrifugation, PCR amplification, and dot blot screening. Among 2000 clones from the subtracted cDNA library, one clone with a full-length HCC-related cDNA was eventually obtained. Sequence analysis of this clone showed it to exhibit 90 and 60% similarity with the rat cysteine sulfinic acid decarboxylase (CSAD) and mammalian glutamic acid decarboxylases (GAD), respectively. Differences between our sequence data on CSAD and those reported previously were observed at two positions, which arose from a single amino acid substitution and frame shift mutation. The CSAD expression was restricted to the liver and kidney of rats. During hepatocarcinogenesis, expression of the CSAD mRNA and its protein was stimulated in the precancerous liver and maintained its high expression afterward. Interestingly, a high level of anti-CSAD autoantibody was detected in the HCC-bearing rats. The titer of anti-CSAD autoantibodies in these rats was 30-200 times higher than that in normal rats. The anti-CSAD autoantibody appeared in the precancerous state and was maintained afterward, and its pattern of appearance was similar to that of CSAD mRNAs and proteins. Thus, we propose that the high-titer CSAD autoantibody resulted from increased CSAD gene expression in the liver due to stimulation by the HCC. These results remind us of human autoimmune diseases including insulin-dependent diabetes mellitus and stiff-man syndrome, which are caused by autoantibodies against GAD.

Amino Acid Sequence↗

PubMed1996

Expression of heme oxygenase and inducible nitric oxide synthase mRNA in human brain tumors.

Heme oxygenase-1, a key enzyme in heme catabolism, and inducible nitric oxide synthase (iNOS) are responsible for production of carbon monoxide and nitric oxide (NO), respectively. Expression of each enzyme has been shown to be modulated by heme and NO, raising a possibility for the coordinated regulation of the two enzymes. We therefore analyzed the expression levels of both mRNA in humans using brain tumors. Either heme oxygenase-1 or iNOS mRNA was expressed at higher levels in brain tumors compared to the brain tissue, but their expression levels were not apparently correlated. In the brain tumor cell lines, treatment with cytokines increased the expression of iNOS mRNA but not heme oxygenase-1 mRNA, whereas treatment with an NO donor increased the expression of heme oxygenase-1 mRNA but not iNOS mRNA. These results suggest the separate regulation of expression of both enzyme mRNA in humans.

Adult↗