Crippling p53 activities via knock-in mutations in mouse models.

The tumor suppressor p53 is the most frequently mutated gene in human cancer. In vivo models have been generated using knock-in alleles in which missense mutations are introduced that mimic the kinds of mutations found in human cancers, or that abolish specific p53 functions. Critically, these studies examine the in vivo and physiological functions of p53. Studies indicate that p53 missense mutations in the DNA-binding domain identical with those inherited in the Li-Fraumeni syndrome, have distinct properties. Studies in mice with mutants that separate cell-cycle arrest and apoptosis functions of p53 show delayed onset of tumor development, suggesting that both p53 functions are crucial for suppressing tumors. Mice with mutations at post-translational modification sites exhibit subtle effects on p53 activity and tumor development, indicating a fine-tuning mechanism of p53 activity in vivo. Importantly, each mutant mouse has a distinct phenotype, suggesting diverse and exquisite mechanisms of p53 regulation in different environments, different tissues and different genetic backgrounds. The generation of these mutant p53 knock-in mice has laid the groundwork for future studies to elucidate the in vivo physiological function of mutant p53 and to examine cooperating effects in combination with other alterations.

EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells.

The translocation t(11;22) is a common chromosomal abnormality detected both in Ewing's sarcoma and in primitive neuroectodermal tumor cells. The translocation results in an EWS-Fli1 fusion gene, made up of the 5' half of the EWS gene on chromosome 22 fused to the 3' half of the Fli1 gene on chromosome 11. Recent studies have evaluated possible roles of the fusion gene products. However, the biological significance of EWS-Fli1 is still unknown. Using a competitive polymerase chain reaction technique, we show here that there might be a correlation between the expression levels of the EWS-Fli1 fusion gene and the proliferative activities of Ewing's sarcoma and primitive neuroectodermal tumor cells. When the EWS-Fli1 expression is inhibited by antisense oligodeoxynucleotides against the fusion RNA, the growth of the tumor cells is significantly reduced both in vitro and in vivo. The data further indicate the growth inhibition of the cells by the antisense sequence might be mediated by G0/G1 block in the cell cycle progression. These results suggest that EWS-Fli1 may play an important role in the proliferation of the tumor cells, and the EWS-Fli1 fusion RNA could be used as a target to inhibit the growth of Ewing's sarcoma and primitive neuroectodermal tumor with the specific antisense oligonucleotide.

Organization and expression of the mouse gene for DNA repair methyltransferase.

06-Methylguanine-DNA methyltransferase (MGMT) is present in various organisms, from bacteria to human cells, and plays an important role in preventing mutations caused by alkylating substances. To understand better the regulatory mechanism involved in the expression of the gene and to construct a mouse model to investigate roles of the enzyme in carcinogenesis, the genomic sequence for mouse methyltransferase was isolated and characterized. The gene consists of 5 exons and spans over 180 kb, whereas mRNA for the enzyme was less than 1 kb. The promoter region for the gene is GC-rich, contains many Sp1 recognition sequences and lacks typical TATA and CCAAT boxes. Primer extension and S1 mapping revealed the existence of multiple transcription initiation sites, among which a major site was defined as +1. The putative promoter region was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and the construct was introduced into mouse NIH-3T3 cells. Deletion analyses revealed that a sequence from -262 to + 56 carries the basic promoter activity. In addition, an adjacent region, spanning from +56 to +95, carries an E2F-like element that greatly stimulates the frequency of transcription. Alteration of TTTTGGGGC to TTAACGGGC considerably reduced the activity.

Dysphagia complications in ankylosing spinal hyperostosis and ossification of the posterior longitudinal ligament. Roentgenographic findings of the developmental process of cervical osteophytes causing dysphagia.

This is the first report of the process of formulation of a cervical osteophyte causing dysphagia. The patient had ankylosing spinal hyperostosis and OPLL and was followed radiographically for a long time before the onset of dysphagia. The radiological observation suggested that dysphagia was produced when the immobile part of the esophagus was compressed by the anterior projecting cervical osteophyte. The immobility of the esophagus is an important factor in determining whether dysphagia occurs. Another possible contributing factor to dysphagia in this patient was the ossification of the cervical anterior and posterior longitudinal ligaments. The OPLL affected intervertebral segmental motion and induced the formation of anterior projecting cervical osteophytes.

Effects of temephos on zooplankton communities in enclosures in a shallow eutrophic lake.

An organophosphorus insecticide, temephos, was applied to large-volume (105 m3) enclosures set up in a shallow eutrophic lake. Application of the chemical at a target concentration of 500 microg litre(-1) eliminated almost all zooplankters. No recovery of cladocerans was evident at the termination of the experiment (47th day after the treatment). Copepods showed a slight recovery after having been absent for 26 days in one enclosure and 40 days in another. The residual chemical remaining in the water until the final day may have suppressed the recovery of the crustacean zooplankters. The rotifer community was reconstructed 16-20 days after the treatment. However, the species composition of this community differed from that of the rotifer community in the control enclosures. Rotifer species might therefore show differences in susceptibility to temephos.

Nutrient dynamics and the eutrophication of shallow lakes Kasumigaura (Japan), Donghu (PR China), and Okeechobee (USA).

We compared the nutrient dynamics of three lakes that have been heavily influenced by point and non-point source pollution and other human activities. The lakes, located in Japan (Lake Kasumigaura), People's Republic of China (Lake Donghu), and the USA (Lake Okeechobee), all are relatively large (> 30 km2), very shallow (< 4 m mean depth), and eutrophic. In all three lakes we found strong interactions among the sediments, water column, and human activities. Important processes affecting nutrient dynamics included nitrogen fixation, light limitation due to resuspended sediments, and intense grazing on algae by cultured fish. As a result of these complex interactions, simple empirical models developed to predict in-lake responses of total phosphorus and algal biomass to external nutrient loads must be used with caution. While published models may provide 'good' results, in terms of model output matching actual data, this may not be due to accurate representation of lake processes in the models. The variable nutrient dynamics that we observed among the three study lakes appears to be typical for shallow lake systems. This indicates that a greater reliance on lake-specific research may be required for effective management, and a lesser role of inter-lake generalization than is possible for deeper, dimictic lake systems. Furthermore, accurate predictions of management impacts in shallow eutrophic lakes may require the use of relatively complex deterministic modeling tools.

Residual pesticides and their toxicity to freshwater shrimp in the littoral and pelagic zones of Lake Kasumigaura, Japan.

Pesticide residues in water and their toxicity to the freshwater shrimp (Paratya compressa improvisa) were studied in a river mouth in Takahamairi Bay, Lake Kasumigaura, from April to July in 1987. Concentrations of fenthion, diazinon and fenobucarb in water of the littoral zone were lower than that in the pelagic zone. The maximum concentration of fenthion, diazinon, iprobenfos and simetryn in water were 1.9, 0.8, 6.5, and 1.1 micrograms 1(-1), respectively. The 4-day mortality of the freshwater shrimp increased in lake water at Takahamairi Bay, reaching 50% at maximum in May. The mortality was probably due to residual insectside fenthion.

[Three cases of brain injury due to windshield impact (author's transl)].

Three cases of brain injury due to windshield impact (tempered windshield) were reported. In the case of brain injury, multitude of small cracks injured the frontal lobe through the orbital roof with facial injuries due to windshield impact. Though we are apt to misdiagnose because of very small wounds, the diagnosis can be made easily from the plain film. The craniotomy, with complete removal of glass fragments and dural plasty, must be performed for the purpose of preventing infection and cerebrospinal fluid rhinorrhea. We believe that the use of seat belt and H.P.R. type laminated windshield is the best precaution against the windshield impact.

[Facial nerve reconstruction--after the operation of C-P angle tumor].

Nineteen patients who undergo facial nerve reconstruction after the operation of cerebellopontine angle tumor from 1964 to 1981 were investigated. Eighteen cases were of acoustic neurinoma, and one was of low grade astrocytoma. Spinal accessory-facial nerve anastomosis was performed in thirteen cases. Cross facial nerve graft was done in three cases. Hypoglossal-facial nerve anastomosis, phrenico-facial nerve anastomosis, and intracranial direct anastomosis were done in one case each. In spinal accessory-facial nerve anastomosis cases, good result was obtained only in 30%, but using microsurgical technique since 1972, its rate went up to 50%. In cross facial nerve anastomosis cases in which two sural nerve grafts were used and the zygomatic and the buccal branches of the right and left connected each other, only one of three revealed good result. The cases of hypoglossal and intracranial direct facial anstomosis resulted in good recovery. As our conclusion, it is difficult to obtain the powerful reinnervation by means of the spinal accessory facial nerve anatomosis and the cross facial nerve graft. Therefore, the best method to be chosen in facial nerve reconstruction seems intracranial direct anastomosis. If the method is impossible, hypoglossal-facial nerve anastomosis should be chosen as the second best. The cross facial nerve graft seems to be leaving much room for technical improvement.

[Evaluation of radiation immunochemotherapy in the treatment of malignant glioma. Combined use of ACNU, VCR and PS-K].

In a follow up study of 38 patients with supratentrial malignant glioma verified histologically during the 3 years from 1979 to 1982, the same therapeutic method which was the postoperative synchronized radiation-immunochemotherapy was applied. And we investigated the relationships between the survival rate and the histological malignancy, the operative area, and age of admission. Total dose of 5000 to 6000 rad radiation was given after surgery. 0.02 mg/kg of VCR was administered intravenously on the first and the 29th day of radiation, and 2 mg/kg of ACNU was administered intravenously 24 hours after VCR administration. After synchronized radiotherapy, 2 mg/kg of ACNU was given every 6 weeks and 3 g of PS-K was given orally every day. Dose of PS-K was increased especially during the radiation and for 2 weeks after ACNU administration. This radioimmunochemotherapy was applied to 38 patients with malignant glioma, 25 cases of glioblastoma multiforme, 12 cases of malignant astrocytoma, one cases of malignant ependymoma, one case of malignant oligodendroglioma. A complete clinical course of all patients was observed. 18 of 38 cases are surviving. The survival rate of malignant gliomas was 71.2% for one year, 47.6% for 2 years, 34.8% for 3 years. The survival rate of glioblastoma was 56.3% for one year, 36.9% for 2 years, 12.3% for 3 years. The survival rate of the patients receiving macroscopically total removal was higher than that of the patients receiving subtotal removal. The survival rate of the younger patients (under 49 years old) was higher than that of the older patients (over 50 years old). Side effect of this therapy was myelosupression in 75.8%.

MTBP suppresses cell migration and filopodia formation by inhibiting ACTN4.

Murine double minute (MDM2) binding protein (MTBP) has been implicated in cancer progression. Here, we demonstrate one mechanism by which MTBP inhibits cancer metastasis. Overexpression of MTBP in human osteosarcoma cell lines lacking wild-type p53 did not alter primary tumor growth in mice, but significantly inhibited metastases. MTBP downregulation increased the migratory potential of MDM2(-/-)p53(-/-) mouse embryonic fibroblasts, suggesting that MTBP inhibited cell migration independently of the Mdm2-p53 pathway. Co-immunoprecipitation and mass spectrometric analysis identified alpha-actinin-4 (ACTN4) as an MTBP-interacting protein. Endogenous MTBP interacted with and partially colocalized with ACTN4. MTBP overexpression inhibited cell migration and filopodia formation mediated by ACTN4. Increased cell migration by MTBP downregulation was inhibited by concomitant downregulation of ACTN4. MTBP also inhibited ACTN4-mediated F-actin bundling. We furthermore demonstrated that nuclear localization of MTBP was dispensable for inhibiting ACTN4-mediated cell migration and filopodia formation. Thus, MTBP suppresses cell migration, at least partially, by inhibiting ACTN4 function. Our study not only provides a mechanism of metastasis suppression by MTBP, but also suggests MTBP as a potential biomarker for cancer progression.

IL-7Rα deficiency in p53null mice exacerbates thymocyte telomere erosion and lymphomagenesis.

Interleukin-7 (IL-7) is an essential T-cell survival cytokine. IL-7 receptor (IL-7Rα) deficiency severely impairs T-cell development due to substantial apoptosis. We hypothesized that IL-7Rα(null)-induced apoptosis is partially contributed by an elevated p53 activity. To investigate the genetic association of IL-7/IL-7Rα signaling with the p53 pathway, we generated IL-7Rα(null)p53(null) (DKO) mice. DKO mice exhibited a marked reduction of apoptosis in developing T cells and an augmented thymic lymphomagenesis with telomere erosions and exacerbated chromosomal anomalies, including chromosome duplications, breaks, and translocations. In particular, Robertsonian translocations, in which telocentric chromosomes fuse at the centromeric region, and a complete loss of telomeres at the fusion site occurred frequently in DKO thymic lymphomas. Cellular and molecular investigations revealed that IL-7/IL-7Rα signaling withdrawal diminished the protein synthesis of protection of telomere 1 (POT1), a subunit of telomere protective complex shelterin, leading to telomere erosion and the activation of the p53 pathway. Blockade of IL-7/IL-7Rα signaling in IL-7-dependent p53(null) cells reduced POT1 expression and caused telomere and chromosome abnormalities similar to those observed in DKO lymphomas. This study underscores a novel function of IL-7/IL-7Rα during T-cell development in regulating telomere integrity via POT1 expression and provides new insights into cytokine-mediated survival signals and T-cell lymphomagenesis.

MTBP plays a crucial role in mitotic progression and chromosome segregation.

Murine double minute 2 (MDM2) binding protein (MTBP) has been implicated in tumor cell proliferation, but the underlying mechanisms remain unclear. The results of MTBP expression analysis during cell cycle progression demonstrated that MTBP protein was rapidly degraded during mitosis. Immunofluorescence studies revealed that a portion of MTBP was localized at the kinetochores during prometaphase. MTBP overexpression delayed mitotic progression from nuclear envelope breakdown (NEB) to anaphase onset and induced abnormal chromosome segregation such as lagging chromosomes, chromosome bridges, and multipolar chromosome segregation. Conversely, MTBP downmodulation caused an abbreviated metaphase and insufficient mitotic arrest, resulting in abnormal chromosome segregation, aneuploidy, decreased cell proliferation, senescence, and cell death, similar to that of Mad2 (mitotic arrest-deficient 2) downmodulation. Furthermore, MTBP downmodulation inhibited the accumulation of Mad1 and Mad2, but not BubR1 (budding uninhibited by benzimidazoles related 1), on the kinetochores, whereas MTBP overexpression inhibited the release of Mad2 from the metaphase kinetochores. These results may imply that MTBP has an important role in recruiting and/or retaining the Mad1/Mad2 complex at the kinetochores during prometaphase, but its degradation is required for silencing the mitotic checkpoint. Together, this study indicates that MTBP has a crucial role in proper mitotic progression and faithful chromosome segregation, providing new insights into regulation of the mitotic checkpoint.

A deficiency in Mdm2 binding protein inhibits Myc-induced B-cell proliferation and lymphomagenesis.

Mdm2 binding protein (MTBP) has been implicated in cell-cycle arrest and the Mdm2/p53 tumor suppressor pathway through its interaction with Mdm2. To determine the function of MTBP in tumorigenesis and its potential role in the Mdm2/p53 pathway, we crossed Mtbp-deficient mice to Emu-myc transgenic mice, in which overexpression of the oncogene c-Myc induces B-cell lymphomas primarily through inactivation of the Mdm2/p53 pathway. We report that Myc-induced B-cell lymphoma development in Mtbp heterozygous mice was profoundly delayed. Surprisingly, reduced levels of Mtbp did not lead to an increase in B-cell apoptosis or affect Mdm2. Instead, an Mtbp deficiency inhibited Myc-induced proliferation and the upregulation of Myc target genes necessary for cell growth. Consistent with a role in proliferation, Mtbp expression was induced by Myc and other factors that promote cell-cycle progression and was elevated in lymphomas from humans and mice. Therefore, Mtbp functioned independent of Mdm2 and was a limiting factor for the proliferative and transforming functions of Myc. Thus, Mtbp is a previously unrecognized regulator of Myc-induced tumorigenesis.