Characteristics of NaF-induced differentiation of HL-60 cells.

Sodium fluoride (NaF) is known to stimulate osteoblastic bone formation, but little attention has been given to the possibility that NaF also affects bone resorption and the differentiation of osteoclastic progenitor cells. When human promyelocytic HL-60 cells were treated with NaF (0.5 mM, 0-4 days), cell proliferation was inhibited, and the addition of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10nM, 0-4 days) augmented this antiproliferative effect. NaF increased cellular reduction of nitroblue tetrazolium (NBT), and this effect was strongly augmented by 1,25(OH)2D3. In addition, NaF produced marked changes in cellular morphology, increased cellular adhesion to plastic, reduced the nuclear/cytoplasmic ratio, and increased cellular expression of chloroacetate esterase, but failed to alter cellular nonspecific esterase activity. Furthermore, NaF increased expression of CD11b and CD66b, and this stimulation was enhanced by adding 1,25(OH)2D3. The sum of these changes in classical promyelocytic cellular indices suggest: (1) that NaF stimulates the early stages of HL-60 differentiation toward a granulocyte-like cell and (2) that 1,25(OH)2D3 promotes these actions of NaF. Additional experiments aimed at further understanding the NaF-induced conversion of HL-60 cells identified further changes. NaF also increased cellular production of prostaglandin E2 (PGE2) and nitric oxide (NO) and induced expression of inducible nitric oxide synthase (iNOS); 1,25(OH)2D3 once again augmented these NaF-induced effects. Similarly, NaF stimulated the production of interleukin 1 alpha (IL-1 alpha), IL-6, and tumor necrosis factor-alpha, and 1,25(OH)2D3 again strongly enhanced these effects. Indomethacin completely blocked stimulation of NBT reduction, NO production, and iNOS expression induced by NaF plus 1,25(OH)2D3; adding exogenous PGE2 (0.1-10 ng/ml) to these indomethacin-blocked cultures dose-dependently restored NO production. These additional findings together with the observed slow onset (24-48 h) of NaF and 1,25(OH)2D3 interaction strongly suggest that 1,25(OH)2D3 acts as a cofactor with NaF primarily through interaction with an endogenous NaF-induced cyclo-oxygenase product(s), quite possibly PGE2 itself. Such a mechanism for NaF and 1,25(OH)2D3 interaction would be strongly analogous to the interaction we have recently demonstrated between 1,25(OH)2D3 and PGE1 on the differentiation of HL-60 cells.

srb: a Bacillus subtilis gene encoding a homologue of the alpha-subunit of the mammalian signal recognition particle receptor.

We cloned a Bacillus subtilis gene (srb) encoding a homologue of the mammalian signal recognition particle receptor alpha-subunit (SR alpha). The gene is 987 bp in length and encodes a 329-amino acid protein. The deduced amino acid sequence of the protein shared 26.6, 36.2 and 49.7% identity with those of mammalian SR alpha, archaebacterial DP alpha and Escherichia coli FtsY, respectively. The protein contains three conserved GTP-binding elements like the other three SRP receptor proteins, though the N-terminal portion of the putative B. subtilis protein was shorter than the others. Secondary structure prediction showed than an amphipathic alpha-helix is positioned in the N-terminal region. A defect in srb inhibited cell growth and protein translocation.

The effect of Srb, a homologue of the mammalian SRP receptor alpha-subunit, on Bacillus subtilis growth and protein translocation.

To determine the signal recognition particle (SRP)-SRP receptor (Srb) system in Bacillus subtilis (Bs), we cloned the Bs srb gene, which encodes a homologue of the mammalian SRP receptor alpha-subunit [Oguro et al., DNA Res. 2 (1995) 95-100]. We sequenced a 6098-bp DNA containing srb and analyzed the gene organization. Primer extension experiment and Northern blot analysis revealed that srb constitutes an operon with two additional ORFs. A database search of known proteins revealed that one encodes a homologue of Escherichia coli RNase III [36.0% identical amino acids (aa)] and the other encodes a homologue of yeast Smc1 (26.6% identical aa). We then constructed a Bs mutant in which srb expression was induced by IPTG. The depletion of Srb caused a defect in the cell growth and the cells became filamentous and twisted. Furthermore, pulse-chase experiments using this mutant revealed that the 17% of the beta-lactamase precursor accumulated in the cell after a 4-min chase in the absence of IPTG, although almost all of the precursors were converted into the mature from after a 1-min chase in the presence of IPTG.

Determination of the spleen-blood partition coefficient for water with oxygen-15-water and oxygen-15-carbon dioxide dynamic PET steady-state methods.

METHODS: Regional splenic blood flow (SBF) was quantified by PET using a steady-state method with 15O-carbon dioxide. SBFs were determined using 104 tomographic planes obtained from 49 patients. RESULTS: When the spleen-blood partition coefficient for water (p) was > or = 0.85, significant correlations (p < 0.005) were found between SBF values determined by the steady-state and dynamic methods. The best correlation between SBFs determined by the two methods (r = 0.571) was found when p = 0.93. The best regression line, however, was thought to be the line when p = 0.93. The regression line between SBF calculated by the steady-state method (y) and SBF determined by the dynamic method (x) was y = 0.57 x + 0.03 with an F ratio of 48.75 (d.f. = 103, p = 5.0 x 10(-8)%, by ANOVA) when p = 0.92. CONCLUSION: A quick evaluation of SBF can be made by using the newly defined regression line.

A truncated Bacillus subtilis SecA protein consisting of the N-terminal 234 amino acid residues forms a complex with Escherichia coli SecA51(ts) protein and complements the protein translocation defect of the secA51 mutant.

Although wild-type Bacillus subtilis SecA barely complements the growth and protein translocation defect of Escherichia coli secA51(ts) at the non-permissive temperature, an N-terminal peptide of B. subtilis SecA complements the defects. To elucidate the mechanism of this complementation, a series of plasmids encoding truncated SecA proteins was constructed and their products were analyzed in E. coli cells. The truncated B. subtilis SecA protein consisting of the N-terminal 234 amino acid residues (BN234) complemented the growth and protein translocation defects of E. coli secA51 but not those of another secA amber mutant, E. coli secA13(ts). BN234 existed in both a soluble form, possibly as a homodimer, and a higher-molecular-weight complex in E. coli strain MM52 (secA51). The purified complex, consisting of at least BN234, SecA51, and ATP-dependent protease La, was held together by a cross-linking reagent, EDAC. The other truncated proteins consisting of the N-terminal 584 or 396 amino acid residues and the C-terminal 607 residues of B. subtilis SecA did not complement the two E. coli mutants or form a complex with SecA51. These results suggest that BN234 and SecA51 proteins form a functional complex in vivo and complement the defects of E. coli MM52.

Possible regulation of epidermal growth factor-receptor tyrosine autophosphorylation by calcium and G proteins in chemically permeabilized rat UMR106 cells.

A model using chemically permeabilized cells was developed to examine mechanisms that regulate protein tyrosine phosphorylation in osteoblastic cells. Using either permeabilized UMR106 osteoblastic or A431 (reference) cells, epidermal growth factor (EGF)-induced cellular tyrosine phosphorylation, and whether there are previously unrecognized interactions between this transduction pathway and Ca2+- or G protein-dependent signalling pathways, were investigated. Both permeabilized cell types, when maintained in non-supplemented cytoplasmic substitution solution (basic CSS), responded to EGF (1-100 ng/ml) with dose-dependent increases in tyrosine phosphorylation. A complex and time-dependent pattern of phosphotyrosine-containing proteins resulted, but the profile of tyrosine phosphorylated proteins was appreciably less complex than in intact cells. Supplementation of basic CSS with MgATP restored the normal complexity of the profiles for EGF-induced tyrosine phosphorylation proteins in both permeabilized cell lines and produced a more sustained accumulation of phosphoprotein products in A431 cells. Adding Ca2+ (< or = 10(-6) M), with or without exogenous MgATP, dose-dependently attenuated EGF-induced tyrosine phosphorylation of EGF receptors (EGFR) and other substrates in UMR106 cells, but was less effective in A431 cells. In both cell types, genistein, an inhibitor of tyrosine kinases, was more effective in attenuating EGF-induced receptor tyrosine phosphorylation in permeabilized cells. Similarly, orthovanadate, an inhibitor of protein tyrosine phosphatases, stimulated the accumulation of phosphoprotein products more effectively in permeabilized cells. Thus, the permeabilization preserves many features of intact cells while facilitating manipulation of intracellular conditions. NaF reproducibly produced a significant vanadate-like action in permeabilized cells that was somewhat stronger than its effect on intact cells. In contrast, the well-known inhibition of tyrosine phosphorylation by phorbol 12-myristate 13-acetate (PMA) was less effective in permeabilized cells than in intact cells; these actions of PMA were Ca2+-dependent. In addition, guanylyl-imidodiphosphate (Gpp(NH)p) attenuated tyrosine phosphorylation in UMR106 cells, and this effect was specifically blocked by guanosine 5'-O-(2-thiodiphosphate) (GDPbetas). These results strongly suggest that there is crosstalk between EGFR-activated tyrosine phosphorylation/dephosphorylation pathways and both Ca2+- and G protein-mediated pathways in UMR106 cells, revealing a previously unrecognized modulation of EGF signalling in osteoblast-like cells that contrasts with the simpler regulatory mechanisms found in A431 cells.

Reproducibility of repeated human regional splenic blood flow measurements using [15O] water and positron emission tomography.

The reproducibility of measurement of regional splenic blood flow by dynamic positron emission tomography (PET) using [15O]water was evaluated. In 19 patients, the correlation between the first and second of two serial dynamic measurements was significant (P=1.78 x 10(-6); r=0.858). The regression equation was y = 1.06x, and the slope of the line described had a 95% confidence interval of 0.09. The error apparent between the two measurements was 0.129 (95% confidence interval 0.059). The results demonstrated sufficiently good reproducibility for measurements of regional splenic blood flow with PET and [15O]water to suggest use of this method for serial measurements intended to detect change, including drug effects.

Errors in the spleen-blood partition coefficient for water in C15O2 steady-state PET.

The aim of this study was to clarify if the value of 0.93, determined for patients with normal livers, is useful as a pathological spleen-blood partition coefficient for water when the splenic blood flow is quantified by the C15O2 steady-state method. A steady-state PET scan with continuous inhalation of C15O2 and a dynamic PET scan with a H(2)15O bolus injection were performed. From 157 patients, 392 slices were chosen as having planes that encompassed the spleen and provided regions of interest with full signal imaging. A comparison of the results of the steady-state and dynamic methods was performed. When 0.93 was adopted as the spleen-blood partition coefficient for water, an error of about 25% was seen in the splenic blood flow of patients with cirrhosis. When measuring splenic blood flow, the H(2)15O dynamic method is necessary. However, a rough estimate of splenic blood flow is possible by the C15O2 PET steady-state method, if this error is known.

Difference in regional hepatic blood flow in liver segments--non-invasive measurement of regional hepatic arterial and portal blood flow in human by positron emission tomography with H2(15)O.

Organ blood flow can be quantitatively measured by positron emission tomography (PET). As the liver has dual blood supplies, arterial and portal, regional hepatic blood flow had not been measured quantitatively. However, we succeeded in simultaneously measuring both regional hepatic arterial and portal blood flow by PET in non-stressed patients. Mean regional portal hepatic blood flow in patients with normal liver and cirrhotic liver was 57.5 and 36.7 ml/minutes/100 g, respectively. Mean regional arterial blood flow was 42.5 and 30.7 ml/minutes/100 g, respectively. A significant difference between regional portal hepatic blood flows in normal and cirrhotic patients was noted. Mean regional portal hepatic blood flow in the lateral, medial, anterior, and posterior segments of the liver was 29.8, 43.4, 50.0, and 40.9 ml/minutes/100 g, respectively. Mean regional arterial blood flow in each liver segment was 37.6, 30.0, 28.2, and 31.6 ml/minutes/100 g, respectively. A significant difference between regional portal hepatic blood flows in lateral and anterior segment was noted. The p value was less than 0.025 and the 95% confidence interval of the difference between means was from -20.2 to -2.7 ml/minutes/100 g by ANOVA. These results showed that regional hepatic blood flow is not the same in all the liver segments.

Using the spleen for time-delay correction of the input function in measuring hepatic blood flow with oxygen-15 water by dynamic PET.

By the spleen, we calculated a time-delay correction of the input function for quantitation of hepatic blood flow with oxygen-15 water and dynamic positron emission tomography. The time delay (deltat) between the sample site and the spleen was calculated based on nonlinear multiple regression analysis when splenic blood flow was determined. Then hepatic blood flow was quantified by a method using the input function and incorporating deltat, which was assumed to be equal to the time delay between the sample site and the liver. Then hepatic arterial and portal blood flows were estimated separately as well as the delay time for passage within the organs of the portal circulation. The mean coefficient of variation and the mean sum of squares of errors decreased to about 70% when total hepatic blood flow was calculated from the results for regions of interest in three slices of the same liver segment. We concluded that using the spleen for time-delay correction of the input function for measuring hepatic blood flow by this method gave satisfactory results.

Quantification of human splenic blood flow (quantitative measurement of splenic blood flow with H2(15)O and a dynamic state method: 1).

Positron emission tomography (PET) by means of a dynamic state method and H2(15)O was performed to quantify splenic blood flow in 20 patients who had no hepatic functional disorders. Non-linear regression analysis was applied to determine splenic blood flow. In calculating arterial input function for the spleen, our original exponential method was used to facilitate computerization. Mean splenic blood flow per 100 g of spleen (SBF) was 168.0 ml/min/100 g with a standard error (SE) of 12.4 ml/min. The mean spleen-blood partition coefficient for water (rho) was 0.767 with a SE of 0.020. Significant correlations were noted between the values for SBF obtained by the exponential method and linear method in which individual increasing values for arterial 15O concentration were used rectilinearly (r = 0.96, p < 0.005) and also between the values for rho obtained by the two methods (r = 0.95, p < 0.005). In order to validate the application of a one compartment model to an organ with a large blood volume such as the spleen, a further experiment was performed with a water flow model simulating splenic circulation. We succeeded in quantifying regional splenic blood flow by PET. It was thought that the quantification of splenic blood flow by our method would be beneficial in the study of splenic circulation, which is expected to be altered under conditions of portal hypertension, liver dysfunction and shock, etc.

Relationship between liver function and splenic blood flow (quantitative measurement of splenic blood flow with H2(15)O and a dynamic state method: 2).

We measured splenic blood flow in 55 patients by means of quantitative splenic positron emission tomography (PET), a novel, dynamic state method with H2(15)O as a tracer. Twenty-four of the 55 patients suffered from liver cirrhosis (LC), 25 showed no evidence of cirrhosis (NR) and 6 patients were diagnosed as having chronic hepatitis (CH). Splenic blood flow per 100 g weight of the spleen (SBF) was significantly correlated with splenic volume (r = -0.39, p < 0.005). The indocyanine green retention test at 15 min (r = -0.39, p < 0.005) and the hepaplastin test (r = 0.37, p < 0.025) also correlated significantly with SBF. The means and 95% confidence intervals for the LC, CH, and NR groups were 117.5 ml/min/100 g (96.6-138.4), 102.5 ml/min/100 g (60.6-144.4), and 160.3 ml/min/100 g (139.8-180.8), respectively. The differences in SBF between these 3 groups were significant (p < 0.01). We conclude that regional splenic blood flow is not proportionate to splenic volume, although the splenic volume does increase with the progressive chronic changes observed in hepatic diseases.

[Superior mesenteric arterial infusion chemotherapy against intestinal obstruction caused by peritoneal carcinomatosis].

For 4 patients with peritoneal carcinomatosis who had severe abdominal pain and vomiting, intra-arterial infusion chemotherapy, via the superior mesenteric artery was performed. After the treatment, all patients were free of their symptoms and began to eat again. Severe complications such as superior mesenteric arterial thrombosis did not occur. It is concluded that our treatment is clinically useful because the quality of life of these patients at the end stage was improved.

[Recurrence and survival rate of advanced gastric cancer after preoperative cisplatin intra-arterial injection therapy].

In our department, curative operations were performed for 49 patients with resectable advanced gastric cancer from Jan. 1988 to Feb. 1990. Among these patients, preoperative intra-arterial therapy using cisplatin (40 or 60 mg) was done for 17 patients. In this report, recurrence and survival rate of these patients were investigated. Survival rate of patients with preoperative intra-arterial injection therapy 42 months after operation was 56.15%, while that of patients without preoperative intra-arterial injection therapy was 56.62%. There were no significant differences between these two groups. And two peritoneal disseminations and one brain metastasis were seen in patients with preoperative intra-arterial injection therapy (recurrence rate, 17.6%), but no liver metastasis and local recurrence were noted. Seven recurrences were observed in patients without pre-operative intra-arterial CDDP injection therapy (local 3, liver 3, peritoneal dissemination 1, recurrence rate, 21.2%). We have already reported that much platinum accumulated in gastric cancer tissue and regional lymph nodes. This high accumulation of cisplatin can be the reason why no local recurrence was seen in patients with preoperative intra-arterial injection therapy. In conclusion, preoperative intra-arterial injection therapy using cisplatin may be an effective method to prevent postoperative local recurrence of resectable advanced gastric cancer.

[Superior mesenteric arterial infusion chemotherapy in patient with both peritoneal carcinomatosis and liver metastasis].

Experimental study of superior mesenteric arterial infusion chemotherapy was done to evaluate the effect of this therapy on metastatic liver tumor. This treatment has been tried clinically for ileus caused by peritoneal carcinomatosis with good result. Moreover, the infused drug is expected to reach the liver via portal route, just like intraportal administration. This study disclosed that superior mesenteric arterial infusion made for a sufficiently high 5-FU concentration of portal blood but the concentration in the metastatic liver tumor was lower when compared with intravenous infusion. However, because the 5-FU concentration in blood of the aorta was lower than in the intravenous administration group, our method may decrease the unfavorable side effects of anti-cancer drugs.

[Complete remission of liver metastasis from rectal cancer following continuous oral administration of tegafur-uracil after intrahepatic arterial single injection of CDDP and 5-FU].

An intrahepatic arterial injection of CDDP, 5-FU, followed by ten months of oral tegafur-uracil administration (2g/day), induced remission for 3 months or more in a 72-year-old male with rectal cancer and synchronous liver metastasis subsequent to anterior resection of the rectum. Tegafur-uracil showed an excellent anticancer effect against colorectal metastatic liver cancers without loss of QOL because a single-low dose of intraarterial anticancer injection was followed by continuous oral administration of tegafur-uracil, and the chemotherapy could be managed to obtain complete remission of the hepatic lesion.

[Clinical study of drug accumulation in gastric cancer after preoperative intra-arterial EAP II injection therapy].

Preoperative intra-arterial injection therapy using etoposide, epirubicin and carboplatin (EAP II) was done for patients with resectable advanced gastric cancer. Twenty-six patients (14 males and 12 females) were treated. The concentrations of adriamycin (ADM) and platinum (Pt) were measured in cancer tissue, normal mucosa and regional lymph-nodes which were obtained operatively and in sera just before operation. But the concentration of etoposide was not measured, because when we used preoperative intra-arterial injection therapy using etoposide, epirubicin and cisplatin (EAP I), the mean concentration of etoposide was less than the detectable limit in all tissues and in sera. There were no significant differences among the mean concentrations of ADM in all tissues and in sera. And the mean concentration of ADM in cancer tissue was not higher than that of intra-arterial EAP I injection therapy. The mean concentration of platinum in sera was significantly lower than in cancer tissues, normal mucosa and lymph-nodes. And the mean concentrations of platinum in cancer tissues and lymph-nodes were higher than those of intra-arterial CDDP or EAP I injection therapy. It was concluded that preoperative intra-arterial EAP II injection therapy may be an effective method to improve the usefulness of preoperative intra-arterial injection therapy for gastric cancer.

[Intermittent intra-arterial infusion chemotherapy of 5-FU and carboplatin for unresectable liver metastases].

Intermittent intra-arterial infusion chemotherapy (5-FU: 500 mg and carboplatin; 100 mg per week) using an implantable access was performed for 19 patients with unresectable liver metastasis of colon cancer (17 cases) and gastric cancer (2 cases). Survival time ranged from 12 to 641 days, and the average was 281.4 days with 50% survival at 276 days. Of 19 cases, one access was infected, two cases had catheter obstruction, and two cases had nausea and vomiting.

[Recurrence and survival rate of advanced gastric cancer after preoperative intraarterial EAP I injection therapy].

In our department, curative operations were performed for 32 patients with advanced gastric cancer from April 1989 to August 1990. Preoperative intra-arterial injection therapy with etoposide (100 mg), pirarubicin (20 mg) and cisplatin (20 mg) was given 18 patients. Recurrence and survival rate were investigated. The survival rate of patients with preoperative intra-arterial injection therapy 45 months after operation was 59.2%, while that of patients without preoperative intra-arterial injection therapy was 75.8%. There were no significant differences between these two groups. Three lymph node recurrences were seen in patients with preoperative intra-arterial injection therapy (recurrence rate, 16.7%). Four recurrences were observed in patients without preoperative injection therapy (peritoneal dissemination 2, liver 1, local 1; recurrence rate, 28.6%). We earlier reported that preoperative intra-arterial cisplatin (40 or 60 mg) injection therapy may reduce the incidence of lymph node recurrence and liver metastasis but may not be effective to prevent postoperative peritoneal recurrence, while no peritoneal dissemination was observed in patients with preoperative intra-arterial EAP I injection therapy. Thus, it was concluded that further study of combination and dose of anti-cancer drug may improve effectiveness of preoperative intra-arterial injection therapy for gastric cancer.

[A case of complete response by intermittent intra-arterial injection of 5-fluorouracil and carboplatin for liver metastases due to pancreatic cancer].

A 53-year-old woman with multiple liver metastases after pancreato-duodenectomy for pancreas cancer was inserted with a catheter from the left subclavian artery to the proper hepatic artery and an access was placed under the thoracic skin. Using this system, she was injected with 500 mg of 5-fluorouracil and 50 mg of carboplatin once a week. After about 7 months, liver metastases disappeared on ultrasonography and computed tomography scan. The patient is still free from recurrence after about 2 years from the initial chemotherapy. This result suggested that intermittent intra-arterial infusion of 5-fluorouracil and carboplatin is effective for liver metastases of pancreatic cancer, after curative resection of a primary tumor.