The structure of CDK4/cyclin D3 has implications for models of CDK activation.

Cyclin-dependent kinase 4 (CDK4)/cyclin D complexes are expressed early in the G(1) phase of the cell cycle and stimulate the expression of genes required for G(1) progression by phosphorylation of the product of the retinoblastoma gene, pRb. To elaborate the molecular pathway of CDK4 activation and substrate selection we have determined the structure of nonphosphorylated CDK4/cyclin D3. This structure of an authentic CDK/cyclin complex shows that cyclin binding may not be sufficient to drive the CDK active site toward an active conformation. Phosphorylated CDK4/cyclin D3 is active as a pRb kinase and is susceptible to inhibition by p27(Kip1). Unlike CDK2/cyclin A, CDK4/cyclin D3 can be inactivated by treatment with lambda-phosphatase, implying that phosphorylated T172 is accessible to a generic phosphatase while bound to a cyclin. Taken together, these results suggest that the structural mechanism of CDK4/cyclin D3 activation differs markedly from that of previously studied CDK/cyclin complexes.

RB silencing compromises the DNA damage-induced G2/M checkpoint and causes deregulated expression of the ECT2 oncogene.

As alterations in retinoblastoma (RB)/E2F pathway are commonly found in human cancers, the molecular mechanism underlying cell cycle deregulation caused by the mutations in the RB/E2F pathway needs to be investigated extensively. Compared with good understanding of RB/E2F functions in G1-S cell cycle progression, it is not fully understood how an abrogated RB pathway affects the G2-M phase of the cell cycle. Here, we report that disruption of RB accelerated G2-M progression in the presence of DNA damage by elevating the expression of a set of mitotic regulatory genes. We generated RB(+)- and (-)-matched cells using short hairpin RNA. In the RB(-) cells, the G2/M checkpoint mediated by a DNA-damaging agent was over-ridden. With microarray analysis, we found that the expression of key G2-M regulatory genes was upregulated in RB(-) cells. In particular, we demonstrated that the proto-oncogene ECT2 was directly regulated by E2Fs. Furthermore, suppression of ECT2 expression by small interfering RNA in RB(-) cells resulted in cytokinesis arrest, suggesting that RB(-) cells lack the regulation of E2F-mediated cytokinesis. These results indicate that aberrant ECT2 expression, observed in various human tumors, could be the direct result of RB/E2F pathway deficiency, thereby contributing to cell division in cancers.

Transplantation of newborn thymus plus hematopoietic stem cells can rescue supralethally irradiated mice.

We attempted to rescue supralethally irradiated (SLI) mice by transplantation of hematopoietic stem cells (HSCs) plus thymus from variously aged donors (fetus, newborn and adult). Although the transplantations of these kinds of HSCs alone showed a very short survival, newborn liver cells (NLCs) (as the source of HSCs) plus newborn thymus (NT) transplantation markedly improved the survival rate. The transplantation attenuated severe damage in the small intestine, which is one of the major causes of death by SLI. In addition, the donor-derived CD4(+) T cells significantly increased with additional NT transplantation. The production of interleukin (IL)-7 and keratinocyte growth factor, which plays a crucial role in protection against radiation injury in the intestine, was the highest in NT. Finally, SLI mice that had received NLC plus IL-7(-/-) NT transplantation plus IL-7 injection showed improved survival, weight recovery and an elevated number of CD4(+) T cells compared with the mice that had received NLC plus IL-7(-/-) NT or plus IL-7 injection alone. These findings suggest that NLCs plus NT transplantation can rescue SLI mice most effectively, and that high production of IL-7 in NT plays a crucial role with induction of CD4(+) T cells.

Actinobacillus actinomycetemcomitans lipopolysaccharide stimulates collagen phagocytosis by human gingival fibroblasts.

INTRODUCTION: Collagen phagocytosis by fibroblasts is involved in the intracellular pathway related to collagen breakdown in soft connective tissues. The possible role of lipopolysaccharide (LPS) in regulating this fibroblast function has not been elucidated so we investigated the effect of LPS from Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, on collagen phagocytic activity in human gingival fibroblasts and associated regulatory mechanisms. METHODS: LPS pretreatment stimulated binding of collagen-coated beads to cells and, subsequently, their internalization. RESULTS: The LPS-activated collagen phagocytic process was enhanced in the presence of the soluble form of CD14 (sCD14) or LPS-binding protein (LBP), while the LPS/LBP treatment activated Akt and induced actin reorganization. Furthermore, these LPS/LBP-induced effects were partially suppressed by adding phosphatidyl-inositol-3 kinase (PI3K) inhibitors. CONCLUSION: These results suggest that A. actinomycetemcomitans LPS disturbs the homeostasis of collagen metabolism within gingival tissue by facilitating collagen phagocytosis by gingival fibroblasts, and serum sCD14 and LBP positively regulate the action of LPS. In addition, the PI3K/Akt signaling is thought to partially mediate the LPS/LBP-stimulated collagen phagocytic pathway, which may be dependent on actin cytoskeletal rearrangement.

Vitamin K therapy for cortical bone fragility caused by reduced mechanical loading in a child with hemiplegia.

Fractures frequently occur at cortical bone sites in children with cerebral palsy, but there is no established therapy. We previously found that treatment with vitamins D and K increased cortical bone mass in children with severe physical disability, and have hypothesized that vitamin K could play a significant role in pediatric cortical bones under conditions with reduced mechanical loading. In the present case report, we treated a right hemiplegic ambulant eight-year-old boy with oral vitamin K (15 mg per day) for eight months. Cortical bone geometries at mid-diaphyseal sites in bilateral tibiae were evaluated before and after the treatment. The cross-sectional total, bone and marrow areas of non-hemiplegic tibia increased by 8.8%, 7.4% and 12.0%, respectively, while those of hemiplegic tibia changed by 9.0%, 14.9% and -3.4%, respectively. As a result, the polar moment of inertia, an indicator of the resistance to torsion forces, increased by 13.0% in the non-hemiplegic tibia and by 63.7% in the hemiplegic tibia. Vitamin K may restrict cortical bone fragility, caused by reduced mechanical loading, through its actions at the endosteal bone marrow interface. Further studies are needed to confirm these findings and to clarify the mechanisms involved.

Relationship of p53, Bcl-2, Ki-67 index and E-cadherin expression in early invasive breast cancers with comedonecrosis as an accelerated apoptosis.

AIMS: To study the relationship between comedonecrosis formation and morphology, apoptosis, and p53, Bcl-2, Ki-67 index and E-cadherin expression in early invasive breast cancer. EXPERIMENTAL DESIGN: Early invasive breast cancers were first divided into two groups according to the presence (CN+ tumours) or absence (CN- tumours) of comedonecrosis. The histological grade, apoptosis, and expression of E-cadherin, Ki-67, p53 and Bcl-2 in the cancer-affected area, and in normal ducts from the specimen, were then examined. RESULTS: Less tubule and gland formation was seen in CN+ tumours than in CN- tumours, although the histological grade between the groups was not different. During early comedonecrosis, cells undergo apoptosis and subsequent necrosis. p53 was higher in CN+ tumours than in CN- tumours and normal ducts, whereas Bcl-2 was lower in CN+ tumours than in CN- tumours and normal ducts. Both tumours had higher Ki-67 than in normal ducts, but no difference was evident between the tumours. CN+ tumours had slightly higher E-cadherin than that in CN- tumours, but lower than that in normal ducts. The level of comedonecrosis was positively correlated with p53, but inversely correlated with Bcl-2 in all tumours, and p53 and Bcl-2 were inversely correlated with each other. Furthermore, comedonecrosis and p53 were correlated with Ki-67 in CN+ tumours, and Bcl-2 was correlated with Ki-67 in CN- tumours. CONCLUSION: Comedonecrosis may be actively regulated through an apoptotic procedure in massive cancers for their survival and progression, and the above proteins may be associated cooperatively in this process. CN+ and CN- tumours may have opposite proliferative systems under the p53-Bcl-2 pathway.

Possible explanation of radioresistance of glioblastoma in situ.

The response of human glioblastoma to radiation was studied in the logarithmic phase and in the plateau phase of growth, and was compared with those of HeLa S3 irradiated under identical conditions. There was no major difference in the in vitro survival curve parameters between glioblastoma and HeLa in the logarithmic growth phase. However, the surviving fractions for glioblastoma with radiation doses in the range used clinically were higher not only when irradiated in the logarithmic growth phase, but also when subcultured 6 hours after irradiation in the plateau phase, than those for HeLa treated under identical conditions. This suggests that the relatively low cure rate of glioblastoma can be partially explained by the intrinsic radiosensitivity in the logarithmic growth phase and by a high surviving fraction for cells irradiated in the plateau phase.

A novel approach for the development of selective Cdk4 inhibitors: library design based on locations of Cdk4 specific amino acid residues.

Identification of a selective inhibitor for a particular protein kinase without inhibition of other kinases is critical for use as a biological tool or drug. However, this is very difficult because there are hundreds of homologous kinases and their kinase domains including the ATP binding pocket have a common folding pattern. To address this issue, we applied the following structure-based approach for designing selective Cdk4 inhibitors: (1) identification of specifically altered amino acid residues around the ATP binding pocket in Cdk4 by comparison of 390 representative kinases, (2) prediction of appropriate positions to introduce substituents in lead compounds based on the locations of the altered amino acid residues and the binding modes of lead compounds, and (3) library design to interact with the altered amino acid residues supported by de novo design programs. Accordingly, Asp99, Thr102, and Gln98 of Cdk4, which are located in the p16 binding region, were selected as first target residues for specific interactions with Cdk4. Subsequently, the 5-position of the pyrazole ring in the pyrazol-3-ylurea class of lead compound (2a) was predicted to be a suitable position to introduce substituents. We then designed a chemical library of pyrazol-3-ylurea substituted with alkylaminomethyl groups based on the output structures of de novo design programs. Thus we identified a highly selective and potent Cdk4 inhibitor, 15b, substituted with a 5-chloroindan-2-ylaminomethyl group. Compound 15b showed higher selectivity on Cdk4 over those on not only Cdk1/2 (780-fold/190-fold) but also many other kinases (>430-fold) that have been tested thus far. The structural basis for Cdk4 selective inhibition by 15b was analyzed by combining molecular modeling and the X-ray analysis of the Cdk4 mimic Cdk2-inhibitor complex. The results suggest that the hydrogen bond with the carboxyl group of Asp99 and hydrophobic van der Waals contact with the side chains of Thr102 and Gln98 are important. Compound 15b was found to cause cell cycle arrest of the Rb(+) cancer cell line in the G(1) phase, indicating that it is a good biological tool.

92nd Annual Meeting of the American Association for Cancer Research. 24-28 March, 2001, New Orleans, Louisiana, USA.

The 92nd Annual Meeting of the AACR comprised over 5000 abstracts, 12 plenary and award lectures and numerous talks in educational sessions, symposia and mini-symposia. Given the wealth of information presented, we narrowed our coverage to the area of prenyltransferase and protein kinase inhibitors. Many rationally designed drugs are now in clinical trials and exciting results were presented for the Bcr-Abl inhibitor STI-571. The cancer community is beginning to envision new ways to evaluate and administer these well-tolerated drugs which do not fit the traditional anticancer drug profile. There is an emphasis in developing surrogate markers for evaluating the mechanism-based effectiveness as well as identifying off-target toxicities. In addition, there is a large effort in investigating effective drug combinations and the use of these new agents as radiosensitisers. Here we present specific examples of these issues as applied to prenylation and protein kinase inhibitors.

Variable expression on lung cancer cell lines of HLA-A2-binding MAGE-3 peptide recognized by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) specific for HLA-A2-binding MAGE-3 peptide (FLWGPRALV) were generated by repetitive stimulation of PBMC with the peptide in the presence of EBV-transformed B blasts and IL-2. Using these CTL, we investigated the expression of the HLA-A2-binding MAGE-3 peptide on lung cancer cell lines. Of 14 cell lines investigated, 1-87, PC-9, OU-LC-KI, 11-18 and LK87 were derived from HLA-A2 positive patients. But cytofluorometry analysis showed that 1-87, PC-9 and OU-LC-KI, but not 11-18 or LK87 expressed the HLA-A2 antigen. All five cell lines expressed MAGE-3 gene mRNA. Twelve of thirteen CTL lines from two HLA-A2 positive donors showed no cytotoxicity against any of the 14 lung cancer cell lines. CTL line TI-1 showed cytotoxicity against 1-87 but not against any of the other cell lines. Treatment of 1-87 with IFN-gamma greatly augmented the cytotoxicity of TI-1 and induced it in the other 12 CTL lines, confirming the expression of the peptide on 1-87. No cytotoxicity was induced by IFN-gamma treatment of PC-9 or OU-LC-KI. However, PC-9 and OU-LC-KI pulsed with the peptide were killed efficiently by all of the CTL lines, suggesting no expression of the peptide on those cells. A low level of cytotoxicity was induced on 11-18 but not LK87 by IFN-gamma treatment, although expression of the HLA-A2 antigen was not observed by cytofluorometry. These findings showed that expression of the HLA-A2-binding MAGE-3 peptide recognized by CTL was variable on lung cancer cell lines.

An epithelial cell line (KNS-62) derived from a brain metastasis of bronchial squamous cell carcinoma.

A brain metastasis of bronchial squamous cell carcinoma in a patient was cultivated. The cells with epithelium-like characteristics were selected from the primary culture, and were subcultured to establish a cell line. The cultured cells were identified to be neoplastic by means of heterologous transplantation to nude mice, in which tumors developed and reproduced histologic characteristics as were seen in the primary tumor of the patient.

Malonate decarboxylase of Pseudomonas putida is composed of five subunits.

Two different forms of malonate decarboxylase were purified from Pseudomonas putida. The active form was composed of the five different subunits alpha (60 kDa), beta (33 kDa), gamma (28 kDa), delta (13 kDa), and epsilon (30 kDa) and the inactive form was composed of the four subunits lacking the epsilon subunit. The former catalyzed the decarboxylation of malonate to acetate, but the latter could not, although it retained both activities of acetyl-CoA:malonate CoA transferase and malonyl-CoA decarboxylase. The delta subunit of the active form was acylated by the incubation with [2-14C]malonyl-CoA, but the delta subunit of the inactive form was not labeled. From the above results and the N-terminal amino acid sequence analysis, it was concluded that the epsilon subunit was an essential subunit to function as malonyl-CoA:ACP transacylase, which was an indispensable component of the enzyme for the cyclic decarboxylation of malonate.

Neurinoma of the trigeminal root presenting as atypical trigeminal neuralgia: diagnostic values of orbicularis oculi reflex and magnetic resonance imaging. A case report.

A case of trigeminal root neurinoma presenting as atypical trigeminal neuralgia was reported. The orbicularis oculi reflex was absent on the affected side, but reappeared after operation. The demonstration of the tumor was much clearer on magnetic resonance imaging. The usefulness of these electrophysiological and radiological studies in differentiating varied entities of trigeminal neuralgia is stressed.

Differential effects of dopamine antagonists on evoked dopamine release from slices of striatum and nucleus accumbens in rats.

The effects of dopamine-receptor antagonists on electrically-evoked dopamine release were compared in the nucleus accumbens and striatal slices of rats. (-)-Sulpiride induced a concentration-dependent increase in the evoked dopamine release from both regions, the increase in the nucleus accumbens being significantly greater than that in the striatum. Clozapine also increased evoked dopamine release from the nucleus accumbens, but not from the striatum. The haloperidol-induced increase in evoked dopamine release from the nucleus accumbens was less than that from the striatum. These findings indicate that, in terms of dopamine transmission, (-)-sulpiride and clozapine, but not haloperidol, predominantly affect the nucleus accumbens rather than the striatum. We have previously reported that the contribution of D3 receptors to the regulation of dopamine release from dopamine nerve terminals is much greater in the nucleus accumbens than that in the striatum. (-)-Sulpiride and clozapine have relatively higher affinity for D3 receptors than does haloperidol. The regional differences in responsiveness of dopamine release to dopamine antagonists could be due to the different affinities to D2 or D3 receptors of the dopamine antagonists.

Brain metastasis from urachal carcinoma.

A case of brain metastasis from urachal carcinoma is reported. Originally the source of the tumor was unknown. Histologic diagnosis of resected specimen was adenocarcinoma. Later removal of urachal carcinoma with partial resection of the bladder revealed adenocarcinoma identical to that of the brain tumor.

Intramedullary ganglioglioma of spinal cord--case report.

Gangliogliomas occur least commonly in the spinal cord. A case of ganglioglioma of C8-Th1 spinal cord in a 24-year-old male is reported. Magnetic resonance imagings are an important element of diagnosis and treatment.

Cerebellopontine angle meningioma associated with cranial accessory nerve neurinoma--case report.

A 46-year-old female presented with a rare association of cerebellopontine (CP) angle meningioma with accessory nerve neurinoma manifesting as headache, occasional diplopia, speech disturbance, swallowing difficulty, and unsteady gait. Magnetic resonance imaging demonstrated a large tumor in the left CP angle. The tumor was totally removed through a lateral suboccipital approach. During the operation another smaller tumor was found originating from the cranial accessory nerve and was also totally removed. Histological examination found that the larger tumor was a meningotheliomatous meningioma and the smaller an Antoni type A neurinoma. The symptoms were apparently due to the larger tumor. Careful examination of neuroimages is necessary even after the main lesions responsible for the symptoms are identified.

Acute purulent discitis with epidural abscess of the cervical spine in an adult--case report.

A 52-year-old male presented with acute purulent discitis and epidural abscess of the cervical spine manifesting as neck pain and slight fever, followed by sudden onset of quadriparesis. Magnetic resonance (MR) imaging showed a low-signal-intensity area in the C6/7 disc space and epidural space ventral to the spinal cord with peripheral enhancement. Surgical exploration using an anterior approach revealed local discitis and epidural abscess, but no osteomyelitis of the neighboring vertebral bodies. Six months after the decompressive procedure to treat the purulent disc and epidural abscess, he had achieved almost full recovery. Such lesions are rare in adults, but should be considered especially when painful spinal symptoms are associated with fever. Early and definitive diagnosis can be achieved by MR imaging with enhancement.

[Therapeutic problems in aged patients with intracranial aneurysms].

In a series of 404 patients with intracranial saccular aneurysm, 61 were over 65 years of age. Ten of these aged patients had no surgery. The operative mortality in the aged patients was 11.8%. While the mortality rate in patients below 65 years was 6.1%. The main complication following intracranial surgery for aneurysm consisted of cerebral infarction due to vasospasm, hydrocephalus, intracranial hematoma and general complications. The incidence of angiographical vasospasm was 22.6% in the aged patients and 43.6% in the young patients. There is no significant difference in vasospasm following subarachnoid hemorrhage between the aged and young patients. Cerebral infarction occurred in 27.5% of the aged patients and in 24.6% of the young patients. Severe cerebral infarction was found in 92.9% of the aged patients and in 58% of the young patients. Vasospasm resulted in broad cerebral infarction significantly more frequently in the aged patients, but cerebral infarction proved was non-fatal in the aged patients. Ventricular dilatation detected by CT was found in 33.3% of the aged patients and in 24.2% of the young patients. In patients with ventricular dilatation, 82.4% of the aged patients needed ventriculo-peritoneal shunt (V-P shunt). On the other hand, 39.3% of the young patients had V-P shunt. There was no significant difference for ventricular dilatation detected by CT between the aged and young patients. The aged patients depended on V-P shunt significantly more than did the young patients.(ABSTRACT TRUNCATED AT 250 WORDS)

[Bacterial intracranial aneurysm associated with infective endocarditis: a case showing enlargement of aneurysm size].

The authors report a case of bacterial intracranial aneurysm associated with infective endocarditis. A 48-year-old male was admitted on March 26, 1994, with complaints of difficulty in speaking and mild swelling of the right leg following mild fever. On examination he showed motor aphasia and mild weakness of the right upper and lower limbs. Cardiac auscultation revealed a grade 3/6 holosystolic murmur. Laboratory data revealed signs of infection through white blood cell count and CRP. Enterococcus faecalis was isolated from the blood culture at the time of admission. A computerized tomographic (CT) scan and magnetic resonance (MR) imaging showed a round mass with perifocal edema. Angiography revealed an aneurysm from the precentral artery of the left middle cerebral artery. A mycotic aneurysm due to bacterial endocarditis was diagnosed. The patient was treated with high doses of antibiotics. However, angiography 2 weeks after the initial study demonstrated the enlargement of the aneurysm and severe narrowing of the angular artery. On April 19, excision of the aneurysm was performed. Operative findings showed degeneration and thickening of the walls of the aneurysm. After the operation, antibiotic therapy was continued. The patient was asymptomatic upon discharge and has continued to do well. Repeated angiography on September 12 showed no further aneurysm. There is a danger of rupture in mycotic aneurysm due to bacterial endocarditis. It is important to repeat angiography and to manage the primary disease. If an aneurysm enlarges with serial angiography, it should be treated surgically without further delay.