CHIP buffers heterogeneous Bcl-2 expression levels to prevent augmentation of anticancer drug-resistant cell population.

Many types of cancer display heterogeneity in various features, including gene expression and malignant potential. This heterogeneity is associated with drug resistance and cancer progression. Recent studies have shown that the expression of a major protein quality control ubiquitin ligase, carboxyl terminus of Hsc70-interacting protein (CHIP), is negatively correlated with breast cancer clinicopathological stages and poor overall survival. Here we show that CHIP acts as a capacitor of heterogeneous Bcl-2 expression levels and prevents an increase in the anticancer drug-resistant population in breast cancer cells. CHIP knockdown in breast cancer cells increased variation in Bcl-2 expression levels, an antiapoptotic protein, among the cells. Our results also showed that CHIP knockdown increased the proportion of anticancer drug-resistant cells. These findings suggest that CHIP buffers variation in gene expression levels, affecting resistance to anticancer drugs. In single-cell clones derived from breast cancer cell lines, CHIP knockdown did not alter the variation in Bcl-2 expression levels and the proportion of anticancer drug-resistant cells. In contrast, when clonal cells were treated with a mutagen, the variation in Bcl-2 expression levels and proportion of anticancer drug-resistant cells were altered by CHIP knockdown. These results suggest that CHIP masks genetic variations to suppress heterogeneous Bcl-2 expression levels and prevents augmentation of the anticancer drug-resistant population of breast cancer cells. Because genetic variation is a major driver of heterogeneity, our results suggest that the degree of heterogeneity in expression levels is decided by a balance between genetic variation and the buffering capacity of CHIP.

Quiescence and γH2AX in neuroblastoma are regulated by ouabain/Na,K-ATPase.

BACKGROUND: Cellular quiescence is a state of reversible proliferation arrest that is induced by anti-mitogenic signals. The endogenous cardiac glycoside ouabain is a specific ligand of the ubiquitous sodium pump, Na,K-ATPase, also known to regulate cell growth through unknown signalling pathways. METHODS: To investigate the role of ouabain/Na,K-ATPase in uncontrolled neuroblastoma growth we used xenografts, flow cytometry, immunostaining, comet assay, real-time PCR, and electrophysiology after various treatment strategies. RESULTS: The ouabain/Na,K-ATPase complex induced quiescence in malignant neuroblastoma. Tumour growth was reduced by >50% when neuroblastoma cells were xenografted into immune-deficient mice that were fed with ouabain. Ouabain-induced S-G2 phase arrest, activated the DNA-damage response (DDR) pathway marker γH2AX, increased the cell cycle regulator p21(Waf1/Cip1) and upregulated the quiescence-specific transcription factor hairy and enhancer of split1 (HES1), causing neuroblastoma cells to ultimately enter G0. Cells re-entered the cell cycle and resumed proliferation, without showing DNA damage, when ouabain was removed. CONCLUSION: These findings demonstrate a novel action of ouabain/Na,K-ATPase as a regulator of quiescence in neuroblastoma, suggesting that ouabain can be used in chemotherapies to suppress tumour growth and/or arrest cells to increase the therapeutic index in combination therapies.

[True pulmonary carcinosarcoma suspected as a metastatic lung tumor].

This patient, a 53-year-old man, has underwent operation on the diagnosis of esophageal cancer 2 years ago. An abnormal shadow was detected in the left lung field and he was admitted to our hospital for further examination. On the suspicion of metastatic lung tumor by transbronchial brushing cytology, partial resection of the left lower lobe was performed. Histologically the tumor was of carcinomatous (squamous cell carcinoma and adenocarcinoma) and sarcomatous (fibrosarcoma and chondrosarcoma) elements, so the patient was diagnosed as "true" pulmonary carcinosarcoma. True pulmonary carcinosarcoma is one of the very rarest neoplasms of the lung.

Squalene synthase inhibitors reduce plasma triglyceride through a low-density lipoprotein receptor-independent mechanism.

Inhibitors of squalene synthase are considered to be candidate drugs to reduce both plasma cholesterol and triglyceride. However, little is known about the mechanism of squalene synthase inhibitor-specific effect on plasma triglyceride. In this study, we confirmed the triglyceride-lowering effect of ER-27856, a potent squalene synthase inhibitor prodrug, in rhesus monkeys. To determine the role of low-density lipoprotein (LDL) receptor in the triglyceride-lowering effect of squalene synthase inhibitors, we intravenously administered ER-28448, the active form of ER-27856, to Watanabe heritable hyperlipidemic (WHHL) rabbits for 4 days. In heterozygotes, ER-28448 reduced plasma cholesterol and triglyceride by 52% and 37%, respectively. In homozygous rabbits, in contrast, ER-28448 lowered plasma triglyceride by 40% but did not lower plasma cholesterol. Orally administered ER-27856 reduced plasma triglyceride in homozygous animals but atorvastatin and bezafibrate did not. In hepatocytes isolated from homozygous WHHL rabbits, squalene synthase inhibitors but not atorvastatin reduced triglyceride biosynthesis. These data demonstrate that squalene synthase inhibitors reduced plasma triglyceride through an LDL receptor-independent mechanism, which was distinct from that of the triglyceride-lowering action of atorvastatin or bezafibrate. The reduction of hepatic triglyceride biosynthesis may play an important role in the hypotrigyceridemic action of squalene synthase inhibitors.

Expressions of bradykinin B2-receptor, kallikrein and kininogen mRNAs in the heart are altered in pressure-overload cardiac hypertrophy in mice.

Angiotensin-converting enzyme inhibitors prevent cardiac hypertrophy in vivo, and a component of this ameliorative effect has been attributed to accumulation of kinins in cardiac tissues. However, little is known regarding the levels of kallikrein-kinin components in the heart during the development of cardiac hypertrophy. The objectives of the present study were to define the effects of pressure-overload cardiac hypertrophy on cardiac levels of kininogen, kallikrein and bradykinin B2 receptor mRNAs. The pressure-overload induced by aortic constriction produced cardiac hypertrophy in mice after 14 and 28d, assessed from the increased ratios of heart weight to body weight and elevation of brain natriuretic peptide mRNA in the heart. B2 receptor mRNA rapidly decreased in the heart within 7 d after the operation, subsequently returning to those of sham-operated animals. In contrast, levels of both low-molecular-weight kininogen and tissue kallikrein mRNAs were increased 7, 14 and 28 d after aortic constriction. These findings suggest that the mechanical load or stretch in cardiac tissue by pressue-overload rapidly produces the downregulation of B2 receptor expression during the initial stage which may allow the promotion of cardiac hypertrophy induced by a mediation of hypertophic factors such as angiotensin II, while upregulation of kininogen and kallikrein mRNAs during the chronic stage may lead to an enhancement of local kinin generation in the heart, from which further progression of cardiac hypertrophy during later stages may be regulated.

Effect of ER-27856, a novel squalene synthase inhibitor, on plasma cholesterol in rhesus monkeys: comparison with 3-hydroxy-3-methylglutaryl-coa reductase inhibitors.

Squalene synthase (SQS; EC 2.5.1.21) plays an important role in the cholesterol biosynthetic pathway. We discovered ER-28448, 5-(N-[2-butenyl-3-(2-methoxyphenyl)]-N-methylamino)-1, 1-penthylidenebis(phosphonic acid) trisodium salt, as a potent and selective inhibitor of SQS. ER-28448 inhibited SQS in rat liver microsome with an IC(50) value of 3.6 nm. We also prepared ER-27856, the tripivaloyloxymethyl ester prodrug of ER-28448. Although less active than ER-28448 in a liver microsome assay, ER-27856 more potently inhibited cholesterol biosynthesis in rat hepatocytes; and ER-27856 orally inhibited de novo cholesterol biosynthesis in Sprague-Dawley rats, with an ED(50) value of 1.6 mg/kg. In HepG2 cells, ER-27856 upregulated low density lipoprotein receptor activity to 2.1 times that of control. A time-course study indicated that the inhibitory effect of ER-27856 on cholesterol biosynthesis in rats continued for up to 8 h. In a study of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (HMGRIs), atorvastatin actively suppressed cholesterol biosynthesis for 8 h, whereas the effect of pravastatin and simvastatin diminished at 4 and 8 h, respectively. In rhesus monkeys, 4 days of oral administration of ER-27856 lowered plasma total cholesterol (TCHO) more potently than did these HMGRIs. Whereas atorvastatin significantly elevated plasma alanine aminotransferase, a marker of hepatotoxicity, to 3.7 times at 100 mg/kg, ER-27856 increased the level only 1.4 times at 10 mg/kg, at which doses the hypocholesterolemic effect was equivalent. During 28 days of administration, ER-27856 reduced TCHO and non-high density lipoprotein (non-HDL) cholesterol by 72 and 95%, respectively. These results demonstrate that ER-27856 had more potent hypocholesterolemic activity and less hepatotoxic effect than HMGRIs. ER-27856 may contribute to the treatment of hypercholesterolemic patients.

[Pulmonary vein reconstruction for lung carcinoma].

Four cases of primary lung carcinoma (two squamous cell carcinomas and two adenocarcinomas) were performed right middle lobectomy combination with the reconstruction of upper pulmonary vein to remain the upper lobe. Lung carcinomas were in the right lobe and infiltrated to lower margin of upper pulmonary vein in all cases. After resection of the right middle lobe and an affected portion of upper pulmonary vein, the defect of the upper pulmonary vein was replaced with auto-pericardial graft in three cases, and the other one was closed by continuous suture of 5-0 plorene. Pathological classification of these four cases was stage IB in one patient, stage IIB in two and stage IV in one. All patients died from 6 to 53 months after operation (average: 31 months). Metastasis to distant organs was confirmed in all cases, so the prognosis of them was generally poor. Reconstruction of pulmonary vein may be feasible to avoid over resection of other lobes, because pulmonary function can be preserve as well as in the bronchoplasty.

[A case of medullary carcinoma of the thyroid gland: metachronous bilateral metastasis of the neck and mediastinal lymph nodes were treated successfully by staged resection].

This patient, a 52-year-old male, underwent subtotal thyroidectomy on the diagnosis of medullary carcinoma of the thyroid gland in 1980 and postoperative course was uneventful. Since November 1990 he had a persistent diarrhea for 6 months and was admitted to the hospital for the further examination on June 1991. The serum CEA and calcitonine level was very high and chest CT scan findings showed the swelling of right neck and mediastinal lymph nodes. Dissection of the lymph nodes was performed by anterior approach which was gained through a proximal median sternotomy extended into the anterior fourth intercostal space as well as to the base of the neck on the right side. On the pathological examination it was metastasis of medullary carcinoma of the thyroid gland. And 50 months later after second operation he had a persistent diarrhea once again. Left neck and mediastinal lymph node metastasis was detected by chest CT with high serum CEA and calcitonine level. Similarly resection was performed by the same anterior approach on the left side. Irrespective of the extended resection he was free of severe complication; he is still alive 10 months after the third operation without any evidence of recurrence and his current performance status is very good.

[Assessment of surgery for primary lung cancer with dissemination or malignant effusion of pleura (T4 advanced lung cancer)].

Twenty-four cases of primary lung cancer with dissemination or malignant effusion of pleura detected preoperatively or intraoperatively were surgically treated at our hospital. Mean survival time (MST) and two-year survival rate (2 YSR) were analyzed on their resected cases and non-resected cases with similar lesion. MST and 2 YSR of 19 cases with lobectomy, 5 cases with pneumonectomy including pleuropneumonectomy and 15 cases with no surgical procedure were 2.77 +/- 0.60 years, 53.4%, 1.51 +/- 0.50 years, 26.7% and 0.99 +/- 0.15 years, 6.7%. MST and 2 YSR of 13 cases with lymph node dissection under R 1 and 6 cases over R 2 on lobectomy group were 1.99 +/- 0.38 years, 37.5% and 5.66 +/- 1.71 years, 66.7%. These findings suggested that lobectomy with lymph node dissection of R 2 over may be a beneficial treatment of lung cancer with dissemination or malignant effusion of pleura.

[A case of lung adenocarcinoma: detection and resection of the metastatic lesion was managed effectively by serum CEA level].

This patient, a 53-year-old male, has had back pain and an abnormal shadow was detected in the right lung field on December 1989. He was admitted to the hospital for the further examination. On the diagnosis of lung cancer with high serum CEA level operation was performed on February 1990. As a results of pathological examination, histological type was adenocarcinoma and pathological stage was pT3N0M0 stage IIIA. After operation the serum CEA level was decreased immediately but it was gradually increased once again. And then 14 months later right adrenal metastasis was detected by abdominal CT with high serum CEA level and resection was performed. Similarly a solitary lymph node metastasis located in abdomen was detected and resected with high serum CEA level 28 months after second operation. In this case detection and resection of the metastatic lesion was managed effectively by serum CEA level. The patient had a good operative course and is alive 76 months after first operation without any evidence or recurrence.

Anti-atherosclerotic effect of E5324, an inhibitor of acyl-CoA:cholesterol acyltransferase, in Watanabe heritable hyperlipidemic rabbits.

E5324, n-butyl-N'-[2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy]-6- methylphenyl]urea, a novel and potent inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), was evaluated for its anti-atherosclerotic and lipid-lowering effects in Watanabe heritable hyperlipidemic (WHHL) rabbits. At 3 months of age, 40 male WHHL rabbits were divided into 4 groups. The rabbits were fed a standard rabbit chow (control group), or standard rabbit chow containing E5324 (0.1% or 0.02%) or 1% probucol for 16 weeks. Even the high dose of E5324 did not lower the plasma total cholesterol levels throughout the experiment. Probucol slightly reduced the plasma cholesterol levels, and showed anti-atherosclerotic activity, i.e., reductions of atherosclerotic plaque formation and cholesterol content in the aorta. Although E5324 did not lower plasma cholesterol, atherosclerotic plaque formation in the aortic arch and thoracic aorta was reduced (by about 34% and 41%, respectively, at the high dose; P < 0.05). Cholesterol content in the aortic arch and thoracic aorta was also reduced (by about 59% and 62% at the high dose, respectively) compared with the control. These results suggest that E5324 acts directly on the arterial wall through ACAT inhibition, and prevents the progression of atherosclerosis in WHHL rabbits.

[Resection of recurrent lung cancer].

The resected recurrent lung cancer cases were evaluated retrospectively. Out of 1,060 cases who received an operation due to primary lung cancer, 21 (1.8%) had recurrent lung cancer which were resected subsequently. Stage I adenocarcinoma cases were found to be most frequent at the first operation (76%). Out of 21 cases, 7 received completion pneumonectomy, 5 had lobectomy and 8 had limited operation, respectively. The 5-year survival rate after the second operation was 36.6% in all the cases. There was no statistical difference in survival rate between the lobectomy group (including completion pneumonectomy) and the limited operation group. There was statistical difference in survival rate between cases who received a second operation in a time span of 2 or more years after the first operation and the cases who received it in less than 2 years. Good prognosis can be expected after the resection of recurrent lung cancer, but further analysis would be required in evaluation of respiratory function as well as biological malignancy of the tumor.

Effect of the acyl-CoA:cholesterol acyltransferase inhibitor, E5324, on experimental atherosclerosis in rabbits.

E5324, n-butyl-N'-[2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy]-6- methylphenyl]urea, a novel and orally absorbable acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, was evaluated for its antiatherosclerotic and antihyperlipidemic effects in cholesterol-fed hypercholesterolemic rabbits. When administered concurrently with a high-cholesterol (0.5% cholesterol) diet for 12 weeks, E5324 (0.0025%, 0.005% and 0.01% in diet) lowered plasma total cholesterol levels dose-dependently (by about 55%-87% at the end of the experiment compared with the control) and also reduced atherosclerotic plaque formation (about 90% reduction at the highest dose; P < 0.01). In pre-established hypercholesterolemic rabbits, which had been pre-fed a high-cholesterol diet for 8 weeks, E5324 administered in the same diet at a dose of 0.005%, 0.01% or 0.02% for 4 weeks significantly reduced plasma cholesterol levels dose-dependently. Cholesterol content and ACAT activity in the aortic arch were also decreased (by about 72% and 58% at the highest dose, respectively) compared with the control. Another ACAT inhibitor, CI-976, had a similar action, but cholestyramine and probucol (2% and 1% in diet, respectively) lacked anti-atherosclerotic activity in this model. Furthermore, when pre-established hypercholesterolemic rabbits were fed normal rabbit chow diet with or without 0.02% E5324 for 4 weeks, changes in plasma cholesterol levels were similar in both E5324-treated and control groups. On the other hand, E5324 significantly reduced cholesterol content and ACAT activity in the aortic arch (by about 52% and 50%, respectively) compared with the control group. These results indicate that E5324 not only has hypocholesterolemic activity, but also may have a direct effect on the arterial wall in experimental atherosclerosis.

Simple and sensitive quantitation method for mevalonic acid in plasma using gas chromatography/mass spectrometry.

The combination of gas chromatography and mass spectrometry using a polar capillary column and isobutane chemical ionization made it possible to determine mevalonic acid (MVA) as the lactone at subnanogram levels using 1 mL of plasma. The pretreatment procedure consisted of only three steps, namely lactonization, washing with chloroform and liquid/liquid extraction. This simple, rapid and sensitive method, having good precision and accuracy, is useful for evaluating the change of plasma MVA, which is well correlated with whole-body cholesterol biosynthesis.

cDNA cloning and expression of Xenopus sperm-specific basic nuclear protein 5 (SP5) gene.

As part of our continuing program to understand the molecular mechanisms controlling the synthesis of sperm-specific nuclear proteins (SPs1-6) during spermatogenesis in Xenopus, we report here on the isolation of a cDNA clone for SP5, the partial sequencing of the amino acids in the SPs, and the expression of the mRNA for SP5. A cDNA clone (pXSP633) was isolated from a cDNA library, previously prepared from poly (A)+ mRNA obtained from Xenopus round spermatids. Determination of the amino acid sequence of the N-terminal regions of all the SPs(1-6) suggested that pXSP633 encodes SP5, whereas SPs3, 4, and 6 are derived from a second mRNA species, and SPs1 and 2 from a third mRNA species. Thus it seems likely that the six SPs are derived from three different mRNA species. Northern blot analyses of RNA, extracted from primary spermatocytes and round spermatids, was performed with oligonucleotide probes specific for SPs4 and 5 mRNAs. The results showed that whereas both SPs4 and 5 mRNAs are expressed in primary spermatocytes, the amount of SP5 mRNA is only about one-fifth of that of SP4 mRNA. However, both mRNA species undergo a similar size change in the length of their poly (A) tracts during spermatogenesis: the size of the mRNA in cultured round spermatids on day 0 was longer than that in primary spermatocytes, but the size of the mRNA in round spermatids on day 6 was shorter than that in round spermatids on day 0.

[A rare case of intrapulmonary sequestration in left upper lung field diagnosed before surgery].

This case is a 30-year-old male who was indicated abnormal shadow in the left upper lung field by chest X-ray film. The aortography was carried out because pulmonary sequestration was suspected by chest CT-scan etc. From the result, rare pulmonary sequestration in the left upper lung field was diagnosed because patterns with inflow of an abnormal artery from descending aorta into the left upper lung field and perfusion of it into the upper pulmonary vein were observed. The wedge resection was performed. The specimen was slightly hard on the whole, and many cartilages or multiple nodules were observed on the cut surface. Histopathologically, normal pulmonary structure was disappeared, and hyperplasia of the lymphatic nodule was remarkably observed in the interstitium, and cystic wall was covered by ciliated cylindrical epithelium.

Clinicopathological significance of nuclear accumulation of tumor suppressor gene p53 product in primary lung cancer.

Since the nuclear accumulation of p53 protein is known to correspond well with mutation of the p53 tumor suppressor gene, the authors examined 88 primary lung cancer specimens immunohistochemically using anti-p53 mouse monoclonal antibody, pAb1801, and analyzed the relationship between the immunohistochemical results and clinicopathological features. Nuclear localization of p53 protein was found in 43/88 (49%) tumor specimens, but not in the corresponding normal lung tissues. The percentage of cases showing nuclear p53 localization varied according to the histological type. In squamous cell carcinoma, nuclear p53 localization was found in 15/26 (57%), appearing more frequently than in other histologic types. However, no obvious correlation was observed between nuclear p53 localization and patients' age, sex, history of smoking, TNM factor, degree of differentiation, or any other clinicopathological features analyzed. In adenocarcinoma, nuclear p53 localization was found in 20/46 (43%). Incidence of positive cases was significantly correlated with regional lymph node metastasis, distant metastasis, and pathological stage (P less than 0.05). These results indicate that mutation of the p53 tumor suppressor gene plays an important role in the development of primary lung cancer, and that nuclear accumulation of p53 protein is a potential prognostic factor in adenocarcinoma of the lung.

Synthesis of sperm-specific basic nuclear proteins (SPs) in cultured spermatids from Xenopus laevis.

The accumulation and synthesis of sperm-specific basic nuclear proteins (SPs) in Xenopus spermatids in vitro were studied by acid-urea-Triton polyacrylamide gel electrophoresis and fluorography. In synchronous cultures of round spermatids, the amount of SP2 and SP3-5 accumulated almost linearly with time, while that of SP1 remained almost constant. Fluorography showed that round spermatids incorporated [14C]arginine mostly into SP1 and SP3-5, very little into SP2, and none into histones. When [14C]arginine was incorporated into cells for 24 h on Days 0, 3, and 6, followed by immediate extraction of basic nuclear proteins, the SP1 band was detected faintly on Day 0 and the intensity increased to the maximum level by Day 3 and remained constant on Day 6; the SP3-5 bands were first detected on Day 3 and their intensity increased by Day 6. Thus, SP1 and SP3-5 were synthesized differentially during the culture period. When [14C]arginine or [14C]lysine was incorporated into round spermatids on Days 0, 3, and 6 for 15 h and chased for 3-12 days, the intensity of the SP2 band increased significantly, while the intensity of the SP1 band decreased concomitantly. This result indicates that SP2 was processed from a precursor protein which is probably SP1.

Isolation of cDNA for a Xenopus sperm-specific basic nuclear protein (SP4) and evidence for expression of SP4 mRNA in primary spermatocytes.

A cDNA library was prepared in lambda gt 11 from poly(A)+ mRNA isolated from a pure population of Xenopus round spermatids and screened with an antibody against SP3-5 (sperm-specific proteins) of Xenopus sperm. Positive clones were sequenced and an arginine-rich clone, designated pXSP531, was obtained. The 473-nucleotide sequence of pXSP531 contained an open reading frame of 237 nucleotides which was preceded by a 5' untranslated region of 67 nucleotides. The 3' untranslated region contained 149 nucleotides, including a consensus polyadenylation signal (AAATAAAA). Twenty nucleotides of a poly(A) tail was contained in the pXSP531. SP3-5 were separated from each other by reverse-phase chromatography and sequenced. The amino acid sequence of the peptide fragments which were obtained by digestion of SP4 with V8 protease and separated by reverse-phase chromatography was identical to the sequence of the N-terminal 43 and C-terminal 15 amino acids deduced from the nucleotide sequence of pXSP531. This result demonstrates that pXSP531 encodes SP4. Northern hybridization of RNA extracted from primary spermatocytes and round spermatids on Days 0 and 6 with SP4 cDNA probe (pXSP531) showed that SP4 mRNA is present both in primary spermatocytes and in round spermatids as is protamine mRNA in the rainbow trout. The size of the SP4 mRNA in round spermatids on Day 0 was longer by 60 nucleotides compared to that in primary spermatocytes and that in spermatids on Day 6 was shorter by 30 nucleotides compared to that on Day 0. These size differences were due to differences in the length of the poly(A) tracts because digestion of poly(A) with ribonuclease H resulted in the shortening of mRNA to the same size for three stages.