Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts.

INTRODUCTION: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. MATERIALS AND METHODS: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. RESULTS: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. DISCUSSION: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases.

Phenotypic association of spontaneous ovulation and sexual receptivity in virgin females of Drosophila melanogaster mutants.

In Drosophila melanogaster females, mating stimulates ovulation/oviposition and switches off receptivity. To investigate the relationship between ovulation and receptivity, we searched for genetic variants in which ovulation occurs in virgins and characterized their behavioral phenotype. Among a collection of 333 P-element insertion lines, we identified eight lines that showed elevated ovulation in virgins. These females show ovipositor extrusion toward courting males, which is normally observed in mated females. To express the amount of rejection behavior, we defined the extrusion index (EI) as a percentage of time that each female extruded the ovipositor within the total time of being courted. There was a positive correlation between ovulation level and EI, suggesting that the two traits are physiologically associated. Genetic analysis of the variants revealed two regions on the third chromosome responsible for the phenotype.

Growth inhibitory effect of a new camptothecin analog, DX-8951f, on various drug-resistant sublines including BCRP-mediated camptothecin derivative-resistant variants derived from the human lung cancer cell line PC-6.

DX-8951f, a new water-soluble camptothecin (CPT) derivative, has been reported to show potent antitumor effects against various tumors in vitro and in vivo. We further evaluated the cytotoxic effect of DX-8951f against eight drug-resistant sublines derived by stepwise exposure of human oat cell carcinoma PC-6 to various drugs. In paclitaxel-, adriamycin-, vincristine- and etoposide-resistant cells, overexpression of P-glycoprotein (P-gp) and a correlative reduction in drug accumulation and typical drug-sensitivity pattern were confirmed. The etoposide-resistant line with the highest P-gp level was cross-resistant also to SN-38, CPT-11 and topotecan (TPT), but not to 9-aminocamptothecin (9-AC), CPT and DX-8951f. SN-38- and CPT-11-resistant cells, of which topoisomerase I activities and levels were similar to those of the parent cells, showed cross-resistance clearly to TPT, 9-AC and mitoxantrone, but hardly to DX-8951f. In these two resistant sublines, the intracellular topotecan level was significantly lower than that in parental PC-6 and the reduced accumulation was found to be mediated by breast cancer resistant protein (BCRP). The cisplatin-resistant variant, which had a 2-fold increase in glutathione content, showed no cross-resistance and the 5-fluorouracil-resistant variant, which had a 50% decrease in glutathione content, exhibited collateral sensitivity to most of the other anticancer agents including DX-8951f. We concluded that DX-8951f showed a potent cytotoxic effect on various types of drug-resistant cells.

Antiproliferative activity and mechanism of action of DZ-3358, a novel pyrimidinyl pyrazole derivative.

The novel pyrimidinyl pyrazole derivative, 1-[5-methyl-1-(2-pyrimidinyl) -4-pyrazolyl]-3-[4-(3-chlorophenyl)-1-piperazinyl]-1-trans-propene hydrochloride (DZ-3358), was found through random screening to exhibit anti-proliferative activity against human and murine cancer cell lines. DZ-3358 induced the mitotic arrest of P388 murine leukemia cells at 0.4 microgram/ml in a time-dependent manner, and inhibited porcine tubulin polymerization. Furthermore, using immunofluorescence techniques, we observed microtubule formation in NUGC-3 human gastric cancer cells treated with DZ-3358. Microtubule formation was disordered, similar to that which occurred when such cells were treated with colchicine. These findings suggest that the mechanism of the anti-proliferative effect of DZ-3358 is the inhibition of mitosis due to the blocking of tubulin polymerization. The differential pattern of growth inhibitory concentrations of DZ-3358 against a series of cancer cell lines was compared with that of colchicine and vincristine, and no correlation was found with the pattern of these two drugs. DZ-3358 may be useful as a new type of tubulin inhibitor and anti-cancer drug.

Matrix interferences in the analysis of digested biological tissues with inductively coupled plasma-mass spectrometry.

To investigate the physiological roles or toxicity of trace or toxic elements, multielement analysis of limited quantities of samples in the biological tissues is required. Inductively coupled plasma mass spectrometry (ICP-MS) suits this requirement, but spectral and nonspectral interferences are inevitable. We examined correction methods for the nonspectral interferences by analyzing signals of 21 elements in various concentrations of HNO3 as well as five major elements (Na, K, P, Ca, and Cl). Using internal standards, the interferences caused by the major elements were corrected, but the interferences caused by HNO3 were impossible to correct for elements with high ionization potentials. The analytical results using the standard addition method on 14 elements in standard reference materials and fresh brain tissues confirmed the accuracy of this method. Thus, we concluded that the standard addition method is useful to correct for the nonspectral interferences.

Separation of selenium-containing proteins in human and mouse plasma using tandem high-performance liquid chromatography columns coupled with inductively coupled plasma-mass spectrometry.

An analytical method that uses two different high-performance liquid chromatography (HPLC) columns in tandem has been developed that separates three major selenium-containing proteins (albumin, glutathione peroxidase, and selenoprotein P) found in human blood plasma. The first column was a heparin affinity column and the second was a gel filtration column whose outlet was directly connected to an inductively coupled plasma-mass spectrometer. The method successfully separated plasma selenium into the three selenium-containing proteins and revealed the preferential retention of selenium in the form of selenoprotein P in a selenium-deficient human and in selenium-deficient mice. Our results also confirm the results of previous studies that showed a preference for supplemented selenium to be taken up as selenoprotein P in rats. Advantages of the tandem column method are that it allows rapid and convenient analyses of the distribution of plasma selenium, and that it is suitable for stable isotope tracer studies and metal interaction studies.

Synthesis and antitumor activity of novel pyrimidinyl pyrazole derivatives.

Novel pyrimidinyl pyrazole derivatives were synthesized and examined for cytotoxic and antitumor activity. Mannich reaction was employed to construct this scaffold. Among the compounds synthesized, a series of propene derivatives exhibited a potent cytotoxic activity against some tumor cell lines including multidrug resistant cell lines due to the overexpression of P-glycoprotein. The vinyl bond moiety in the scaffold was believed to be required for the cytotoxic activity. Among them, compound 14 g, when administered intraperitoneally, showed potent antitumor activity against the malignant ascites caused by intraperitoneal inoculation of P388 cells in mice. This compound also showed high activity against a solid tumor Meth A mouse fibrosarcoma when administered both intraperitoneally and orally.

Synthesis and antitumor activity of ring A- and F-modified hexacyclic camptothecin analogues.

Nineteen ring A- and F-modified hexacyclic analogues of camptothecin were synthesized by Friedländer condensation of appropriately substituted bicyclic amino ketones with tricyclic ketone and were evaluated for cytotoxicity and topoisomerase I inhibitory activity. Seventeen of the compounds showed cytotoxic effects comparable or superior to those of 7-ethyl-10-hydroxycamptothecin (SN-38) against mouse leukemia P388 and human tumor cell lines HOC-21 and QG-56. Introduction of a compact and inductively electron-withdrawing substituent such as a hydroxy, methoxy, chloro, or fluoro group into position 5 of ring A of the hexacyclic compound remarkably increased the antitumor activity. The potency of topoisomerase I inhibition of these compounds showed good correlation with their cytotoxicity. Among them, the 4-methyl-5-fluoro hexacyclic compound was the most potent of all and was 10 times as active as SN-38 in in vitro antitumor activity.

Synthesis and antitumor activity of novel benzophenone derivatives.

Novel benzophenone derivatives were synthesized and screened for cytotoxic and antitumor activity. Friedel-Crafts condensation was employed to construct the benzophenone skeleton. Among the compounds synthesized, morpholino and thiomorpholino benzophenones 3a-d exhibited potent cytotoxic activity against P388 murine leukemia and PC-6 human lung carcinoma cells in vitro, and compounds 3a, 3c, and 3j, when administered intraperitoneally, showed significant antitumor activity against the malignant ascites caused by intraperitoneal inoculation of P388 cells in mice.

Determination of selenium in the human brain by graphite furnace atomic absorption spectrometry.

For the investigation of neurological disorders, a development of simple and accessible methods for determining selenium in human brain samples is required. We devised a method of determining selenium using graphite furnace atomic absorption spectrometry (GFAAS). An electrodeless discharge lamp provided the sufficient sensitivity to determine brain selenium. The matrix interferences were avoided by using high temperature, a prolonged pyrolysis step, and a palladium matrix modifier. The technique of standard addition was used to evaluate the sample concentrations. The accuracy of the method was confirmed by a bovine liver reference material. The detection limit of selenium was 0.04 ng. The determined selenium concentrations of human brain cortex and white matter were higher than those of putamen (115-155 and 206-222 ng/g wet wt, respectively). These GFAAS values agreed with those obtained by fluorometric analysis (r = 0.91, n = 10). Moreover, the GFAAS values were compatible to those reported by other researchers (99-274 ng/g wet wt), in which selenium concentrations in putamen also tended to be higher than the other two regions. We conclude that GFAAS is useful for selenium analysis in brain samples.

Cytotoxic activities of solanum steroidal glycosides.

Cytotoxic activities of 20 steroidal glycosides obtained from Solanum genera plants were examined against various cell lines to provide new evidence as follows: 1) As regarding the sugar linkage, the glycosides possessing a beta-chacotriosyl moiety were the most effective and the presence of the terminal rhamnosyl residue was required for exhibiting strong activity. 2) As for the aglycone, the glycosides carrying a spirostanol aglycone showed the strongest activity and even an aglycone without a sugar linkage presented considerably strong activity.

Analysis of trace elements in the central nerve tissues with inductively coupled plasma-mass spectrometry.

To investigate the roles of various elements in neurological disorders, multi-element analysis of limited quantities of samples in the central nervous system is required. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis suits this requirement, but spectral and non-spectral interferences are inevitable. We studied correction methods for the non-spectral interferences by analyzing signals of 21 elements in various concentrations of HNO3 as well as 5 major elements (Na, K, P, Ca, and Cl). Using internal standards, the interferences caused by the major elements were corrected, but the interferences caused by HNO3 were impossible to correct for the elements with high ionization potentials. Thus, we decided to use a standard addition method to correct these interferences. The spectral interferences on Mn and Fe rising from HNO3 or major elements were compared with the signals of samples. Although the interferences on Mn were negligible, those on Fe were considerable and careful blank subtraction were needed. We measured concentrations of elements in the spinal cord of 4 controls and a patient with amyotrophic lateral sclerosis (ALS) using the standard addition method. The concentrations of Mn, Se, Fe, and Zn in the controls were nearly the same as previously reported values, whereas Mn concentrations in the ALS patient were higher.

Distribution of selenium in human plasma detected by high performance liquid chromatography-plasma ion source mass spectrometry.

The distribution of selenium in human plasma has been investigated by high performance liquid chromatography (HPLC) connected directly to inductively coupled plasma mass spectrometry (ICP-MS). Human plasma was loaded on to a size exclusion column and eluted with 0.01 M sodium phosphate buffer (pH 7.0) at a flow rate of 0.6 ml/min. Four peaks of selenium were detected in the chromatogram. The first selenium peak was obtained in the void volume. The retention time of the third peak was in accord with that of bovine serum albumin as a standard. The forth peak was thought to be a ghost. The method was applied to identify the chemical form of selenium in blood plasma immediately after intestinal absorption. The chromatographic pattern of selenium in postprandial human plasma was compared with that in fasting plasma. The first and third peaks in the postprandial plasma sample were slightly higher than those in the fasting plasma sample. This finding suggests that absorbed selenium is associated with the high molecular weight fraction and mercaptalbumin in blood plasma.

Microwave digestion using dual PTFE containers for analysis of trace elements in small amounts of biological samples.

The analysis of trace elements in biological samples is essential to extend our knowledge on human health and disease. Inductively coupled plasma mass spectrometry (ICP-MS) makes it possible to simultaneously determine these elements in trace amounts. Before analysis, however, biological samples such as organs and tissues must be liquefied and extra organic materials must be decomposed by acid digestion. We established a method of microwave digestion using dual PTFE containers to minimize the amount of samples. Samples (35-45 mg) of standard reference materials, bovine liver (1577a, NIST) and fish flesh (MA-A-2, IAEA), were weighed in PTFE-PFA vials and a small amount of nitric acid (0.5 ml) was added. The vials were sealed and two PTFE-PFA vials were placed in a PTFE-TFM vessel containing 6 ml of pure water. Then the vessels were placed in a rotor and the samples were digested for 38 min in a microwave oven according to a pre-set program. After the program was completed, the samples were analyzed by ICP-MS. The determined values of elements of the microwave-digested samples matched the certified values of the standard reference materials. Therefore, the digestion using dual containers was successfully applied to small samples.

A new water-soluble camptothecin derivative, DX-8951f, exhibits potent antitumor activity against human tumors in vitro and in vivo.

CPT-11, a semisynthetic derivative of camptothecin, exhibited strong antitumor activity against lymphoma, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, and cervical cancer. CPT-11 is a pro-drug that is converted to an active metabolite, SN-38, in vivo by enzymes such as carboxylesterase. We synthesized a water-soluble and non-pro-drug analog of camptothecin, DX-8951f. It showed both high in vitro potency against a series of 32 malignant cell lines and significant topoisomerase I inhibition. The anti-proliferative activity of DX-8951f, as indicated by the mean GI50 value, was about 6 and 28 times greater than that of SN-38 or SK&F 10486-A (Topotecan), respectively. These three derivatives of camptothecin showed similar patterns of differential response among 32 cell lines, that is, their spectra of in vitro cytotoxicity were almost the same. The antitumor activity of three doses of DX-8951f administered i.v. at 4-day intervals against human gastric adenocarcinoma SC-6 xenografts was greater than that of CPT-11 or SK&F 10486-A. Moreover, it overcame P-glycoprotein-mediated multi-drug resistance. These data suggest that DX-8951f has a high antitumor activity and is a potential therapeutic agent.

Antitumor agents. 7. Synthesis and antitumor activity of novel hexacyclic camptothecin analogues.

Eleven novel hexacyclic and three 7,9-disubstituted pentacyclic derivatives of camptothecin were synthesized and evaluated for in vitro cytotoxic activity against P388, HOC-21, and QG-56 and in vivo antileukemic activity against P388 in mice. Hexacyclic compounds which have an additional 5-, 6-, or 7-membered ring cyclized at positions 7 and 9 of camptothecin were prepared by intramolecular cyclization of pentacyclic camptothecin derivatives or Friedländer condensation of the appropriate bicyclic amino ketone and tricyclic ketone. All of the hexacyclic compounds exhibited compatible or superior activity of 7-ethyl-10-hydroxycamptothecin (SN-38) in in vitro assays without regard to the size or type of the additional ring, and three of six compounds showed more than 300% T/C on in vivo assay. These results suggest that the potency of the hexacyclic ring system is higher than that of the original pentacyclic ring system of camptothecin and that the conformational rigidity of substituents at positions 7 and 9 is favorable for antitumor activity.

Mercury and selenium contents in amyotrophic lateral sclerosis in Hokkaido, the northernmost island of Japan.

We evaluated the pathogenicity of mercury (Hg) and selenium (Se) which are supposed to be one of the risk factors in the development of amyotrophic lateral sclerosis (ALS). Hg and Se contents were measured in plasma, blood cells, scalp hair samples of 21 sporadic ALS patients and 36 controls, who included 19 patients with other neurological diseases, in Hokkaido, the northernmost island of Japan. Hg and Se levels in plasma and blood cells of ALS patients were significantly lower in advanced staged ALS patients than controls. Low Hg and Se contents in ALS, being correlated with their disabilities and nutritional conditions, would rather reflect the disease contracted states than the pathogenic roles in ALS.

Antitumor agents. V. Synthesis and antileukemic activity of E-ring-modified (RS)-camptothecin analogues.

Several E-ring-modified analogues of (RS)-camptothecin were synthesized by total synthesis via Friedländer condensation and evaluated for cytotoxicity and antitumor activity against P388 mouse leukemia cells. Among them, (RS)-20-deoxyamino-7-ethyl-10-methoxycamptothecin (25c) was found to be more active than (RS)-camptothecin (1) in the in vivo assay.