Production of bone-resorbing activity and colony-stimulating activity in vivo and in vitro by a human squamous cell carcinoma associated with hypercalcemia and leukocytosis.

A squamous cell carcinoma of 33-yr-old patient who developed marked leukocytosis and hypercalcemia was transplanted into nude mice in which more marked leukocytosis and hypercalcemia also developed. This tumor (LJC-1-JCK) produced a colony-stimulating factor (CSF) and formed a cyst in the tumor from which a CSF-producing cell line (T3M-1) was established. The CSF causes predominantly formation of granulocytic colonies in addition to macrophage colonies. Bone-resorbing activity (BRA) was detected in the cystic fluid and was eluted as two separate peaks with proteins of an apparent molecular weight of 30,000-50,000 and 10,000-20,000. Colony-stimulating activity (CSA) was eluted at an apparent 30,000 mol wt. The conditioned medium of the T3M-1 cells also contained a BRA with an apparent 14,000 mol wt, whereas CSA eluted at an apparent 30,000 mol wt. PTH, epidermal growth factor, transforming growth factor-alpha, prostaglandin Es, and vitamin D could not account for the powerful BRA. In contrast to CSA, BRA was not inactivated by trypsin and more stable at 70 degrees C. When T3M-1 cells were transplanted into nude mice, marked hypercalcemia developed in addition to granulocytosis. Our findings suggest that the tumor produces and secretes a powerful BRA in vivo and in vitro, which is different from CSA in terms of molecular weight, heat stability, and trypsin treatment. We speculate that the synergistic action of CSF that stimulates macrophage colony formation and recruits osteoclast precursors, and BRA, which stimulates mononuclear phagocytes and/or osteoclasts were responsible for a marked increase in osteoclastic bone resorption and humoral hypercalcemia in the patient.

Hematopoietic stem cell transplantation for 30 patients with primary immunodeficiency diseases: 20 years experience of a single team.

We retrospectively analyzed our results of 30 patients with three distinctive primary immunodeficiency diseases (PIDs)--severe combined immunodeficiency (SCID, n = 11), Wiskott-Aldrich syndrome (WAS, n = 11) and X-linked hyper-immunoglobulin M (IgM) syndrome (XHIM, n = 8)--who underwent hematopoietic SCT (HSCT) during the past 20 years. Until 1995, all donors were HLA-haploidentical relatives with T-cell depletion (TCD) (n = 8). Since 1996, the donors have been HLA-matched related donors (MRD) (n = 8), unrelated BM (UR-BM) (n = 7) and unrelated cord blood (UR-CB) (n = 7). Twenty-seven of 30 patients had various pre-existing infections with or without organ damages before HSCT. Conditioning regimen and GVHD prophylaxis were determined according to disease, donor and pretransplant status. Although one of eight patients transplanted with TCD is alive with full engraftment, the other seven died. On the other hand, 18 of 22 patients transplanted without TCD are alive and well, including six of eight transplanted from MRD, seven of seven from UR-BM and five of seven from UR-CB. All 19 survivors did not require Ig supplementation after HSCT. These results indicate that UR-CBT as well as UR-BMT provides good results for PID comparable to MRD-SCT, and that early diagnosis, HSCT at early stage, careful supportive therapy and monitoring for various pathogens are important for the successful HSCT.

Effect of direct cell-to-cell interaction between the KM-102 clonal human marrow stromal cell line and the HL-60 myeloid leukemic cell line on the differentiation and proliferation of the HL-60 line.

Hematopoietic cellular interaction was investigated in a coculture of the human clonal marrow stromal line, KM-102, and the myeloid leukemia cell line, HL-60. In the coculture, a large number of HL-60 cells remained in the supernatant but some of them became firmly attached to KM-102 cells. The attached HL-60 cells showed little positive reaction in the NBT test when the culture was supplemented with 10(-9) to 10(-7) M 1 alpha, 25-dihydroxyvitamin D3. In contrast, differentiation in the supernatant HL-60 cells was strikingly responsive to the agent in a dose-dependent way. Furthermore, the complete inhibition was observed in the incorporation of [3H]thymidine into the attached HL-60 cells with autoradiography, but 23.6% of the supernatant cells moderately incorporated [3H] thymidine into their nuclei. There was no attachment between HL-60 cells and stromal cells from human thymus and lymph node, or between lymphocytic leukemia cells and marrow stromal cells. These results indicate that there is direct cellular interaction between myeloid leukemic cells and marrow stromal cells which modulates the proliferation and differentiation of the myeloid leukemic cells. This modulation by marrow stromal cells is more strongly affected by this interaction than by exogenously added differentiation-inducing agents. Apparently marrow stromal cells produce a definitive milieu for the proliferation and differentiation of myeloid leukemic cells.

Association of hypercalcemia with tumors producing colony-stimulating factor(s).

Two human malignant tumors, which we previously reported to produce colony-stimulating factors (CSFs), were found to be accompanied by remarkable hypercalcemia. A patient with a CSF-producing lower jaw cancer (squamous cell carcinoma) developed a marked granulocytosis (150,000/microliters) and hypercalcemia (more than 215 mg/dl). The tumor was successfully transplanted into nude mice, which developed marked granulocytosis (300,000/microliters) and hypercalcemia (20 mg/dl). White blood cell and serum calcium concentrations of these mice decreased promptly to normal levels when the tumor was excised. Treatment with prednisolone (1.5 mg/kg) or indomethacin (5 mg/kg) had no effect on the serum calcium level of these mice. Parathyroid hormone or prostaglandin E was not increased in the serum of the mice or in the tumor tissue. However, the mice bearing the tumor excreted extremely large amounts of calcium in their urine, and their bony tissues contained less calcium and phosphorus than controls. Moreover, histology of bony tissues of these nude mice clearly demonstrated the decrease in trabecular tissues and cortical thickness as well as remarkable activation of osteoclasts. Another patient with a CSF-producing bronchogenic squamous cell carcinoma showed mild granulocytosis and hypercalcemia. The biopsied tumor tissue was transplanted into nude mice, which developed marked granulocytosis (300,000/microliters) and also severe hypercalcemia (18 mg/dl). These results suggest the presence of a new syndrome of granulocytosis and hypercalcemia associated with CSF-producing tumors. The causal mechanism of the hypercalcemia was shown to be some humoral factor which activates osteoclasts other than parathyroid hormone. Neither prostaglandins nor osteoclast-activating factor seemed to be the cause of the hypercalcemia.

Engineering and biochemical characterization of the rat microsomal cytochrome P4501A1 fused to ferredoxin and ferredoxin-NADP(+) reductase from plant chloroplasts.

Fusion proteins of rat cytochrome P4501A1 with maize ferredoxin I (Fd) and pea ferredoxin NADP(+) reductase (FNR), the last electron transfer proteins of the photosynthetic channel in plant chloroplasts, were obtained by gene fusion in the yeast expression vector pAAH5N. The encoded fusion proteins P4501A1-Fd, P4501A1-FNR, P4501A1-Fd-FNR and P4501A1-FNR-Fd were produced in microsomes of the yeast Saccharomyces cerevisiae AH22. Enzymatic assays were carried out in vitro with the isolated microsomes. P4501A1-Fd-FNR and P4501A1-FNR-Fd were found to catalyze P450-monooxygenase activities towards 7-ethoxycoumarin and the herbicide chlortoluron. P4501A1-Fd-FNR was the most efficient enzyme as measured in vitro in ferricyanide and cytochrome c reductions, as well as P450-monooxygenase assays. Apparent K(m) and k(cat) of P4501A1-Fd-FNR were 70 microM and 7800 min(-1) for NADPH, 13.2 microM and 51.1 min(-1) for 7-ethoxycoumarin, and 21.3 microM and 23. 8 min(-1) for the herbicide chlortoluron, respectively. Fd in P4501A1-Fd-FNR fusion enzyme was found to be a limiting factor compared to P4501A1 fused to the yeast NADPH-cytochrome P450 reductase, an artificial enzyme described previously. The efficiency of electron transfer in the P4501A1 fusion proteins and a possible in vivo molecular coupling of Fd and FNR with microsomal cytochrome P4501A1 produced in plant chloroplasts are discussed.

Purification and immunochemical characteristics of NADPH-cytochrome P-450 oxidoreductase from tobacco cultured cells.

NADPH-cytochrome P-450 oxidoreductase (EC 1.6.2.4) was purified from the microsomal fraction of tobacco (Nicotiana tabacum) BY2 cells by chromatography on two anion-exchange columns and 2',5' ADP-Sepharose 4B column. The purified enzyme showed a single protein band with a molecular weight of 79 kDa on SDS-PAGE and exhibited a typical flavoprotein redox spectrum, indicating the presence of an equimolar quantity of FAD and FMN. This enzyme followed Michaelis-Menten Kinetics with Km values of 24 microM for NADPH and 16 microM for cytochrome c. An in vitro reconstituted system of the purified reductase with a partially purified tobacco cytochrome P-450 preparation showed the cinnamic acid 4-hydroxylase activity at the rate of 14 pmol min-1 nmol-1 P-450 protein and with a purified rabbit P-4502C14 catalyzed N-demethylation of aminopyrine at the rate of 6 pmol min-1 nmol-1 P-450 protein. Polyclonal antibodies raised against the purified reductase reacted with tobacco reductase but not with yeast reductase on Western blot analysis. Anti-yeast reductase antibodies did not react with the tobacco reductase. This result indicate that the tobacco reductase was immunochemically different from the yeast reductase. The anti-tobacco reductase antibodies totally inhibited the tobacco reductase activity, but not the yeast reductase. Also, Western blot analyses using the anti-tobacco reductase antibodies revealed that leaves, roots and shoots of Nicotiana tabacum plants contained an equal amount of the reductase protein. From these results, it was suggested that there are different antibody binding sites, which certainly participate in enzyme activity, between tobacco and yeast reductase.

Cloning and expression in Escherichia coli and Saccharomyces cerevisiae of a novel tobacco cytochrome P-450-like cDNA.

A cDNA library constructed from poly(A)+ RNA of tobacco BY2 cells treated with 2,4-dichlorophenoxyacetic acid was screened by using a synthetic oligonucleotide corresponding to the heme binding region of avocado CYP71A1. A cloned 2-kb cDNA designated as cTBP contained an open reading frame of 1593 bp encoding a protein of molecular size of 58916. The deduced amino acid sequence included a cysteine residue corresponding to fifth ligand of heme-Fe at 497th. The coding sequence was expressed under the control of tac promoter and rrnB terminator in Escherichia coli to yield 7 to 10 nmol P450 equivalent per litre of the culture in the presence of delta-aminolevulinic acid. The modified coding sequences in which NH2-terminal residues 2-25 were replaced by the NH2-terminal 18 amino acid residues of microsomal bovine CYP17 were also expressed under the control of ADH promoter and terminator in Saccharomyces cerevisiae to yield 29 and 30 pmol of P450 equivalent/mg protein in the microsomal fraction, respectively. On co-expression of each of the modified coding sequences and yeast NADPH-cytochrome P-450 oxidoreductase gene, the yeast microsomes exhibited 7-ethoxycoumarin O-deethylase activity. Based on these results, tobacco cTBP was found to encode a novel P450-like species with a monooxygenese activity related to xenobiotic metabolism.

Biphasic kinetic behavior of rat cytochrome P-4501A1-dependent monooxygenation in recombinant yeast microsomes.

Rat cytochrome P-4501A1-dependent monooxygenase activities were examined in detail using recombinant yeast microsomes containing rat cytochrome P-4501A1 and yeast NADPH-P-450 reductase. On 7-ethoxycoumarin, which is one of the most popular substrates of P-4501A1, the relationship between the initial velocity (v) and the substrate concentration ([S]) exhibited non-linear Michaelis-Menten kinetics. Hanes-Woolf plots ([S]/v vs. [S]) clearly showed a biphasic kinetic behavior. Aminopyrine N-demethylation also showed a biphasic kinetics. The regression analyses on the basis of the two-substrate binding model proposed by Korzekwa et al. (Biochemistry 37 (1998) 4137-4147) strongly suggest the presence of the two substrate-binding sites in P-4501A1 molecules for those substrates. An Arrhenius plot with high 7-ethoxycoumarin concentration showed a breakpoint at around 28 degrees C probably due to the change of the rate-limiting step of P-4501A1-dependent 7-ethoxycoumarin O-deethylation. However, the addition of 30% glycerol to the reaction mixture prevented observation of the breakpoint. The methanol used as a solvent of 7-ethoxycoumarin was found to be a non-competitive inhibitor. Based on the inhibition kinetics, the real V(max) value in the absence of methanol was calculated. These results strongly suggest that the recombinant yeast microsomal membrane containing a single P-450 isoform and yeast NADPH-P-450 reductase is quite useful for kinetic studies on P-450-dependent monooxygenation including an exact evaluation of inhibitory effects of organic solvents.

Differential lipid specificities of the repeated domains of annexin IV.

The roles of the four domains of annexin IV in binding to phospholipids and glycolipids were assessed by analyzing the binding of a group of mutant annexins IV in which one or more of the four domains was inactivated by replacing a critical amino residue(s) (Asp or Glu) with the neutral residue Ala. The data reveal that individual annexin domains may have characteristic affinities for different lipids. In particular, inactivation of the fourth domain inhibits the binding to phosphatidylserine (PS) and phosphatidylinositol (PI) but not to phosphatidylglycerol (PG), suggesting that this domain specifically can accommodate the larger head groups of PS and PI whereas the other three domains may form more restricted binding pockets. In order to block binding to PG, domain 1, or both domains 2 and 3 must be inactivated in addition to domain 4, suggesting that all four domains may be able to accommodate the headgroup of PG to some extent. Binding to acidic glycolipids (sulfatides) was also sensitive to inactivation of domain 4. However, in the case of sulfatides the nature of the binding reaction is fundamentally different compared with the binding to phospholipids since the interaction with sulfatides was highly sensitive to an increase in ionic strength. The binding to sulfatides may depend therefore on charge-charge interactions whereas the binding to phospholipid may involve a more specific interaction between the lipid headgroup and the protein surface, and/or interaction of the protein with the hydrophobic portion of a lipid bilayer.

Novel functions and cellular interaction of human lymphoid stromal cells with lymphoid cell lines in vitro.

Lymphoid stromal (SG) cells have been isolated from the lymph node of a patient with malignant lymphoma, and characterized by positive reaction with a monoclonal antibody against the T-zone stromal cells in human lymph nodes. B-acute lymphoblastic leukemia (BALL) cells showed prominent emperipolesis toward SG cells when they were cocultured, whereas T-acute lymphoblastic leukemia (TALL) cells attached firmly to the surface of SG cells. Autologous peripheral B and T cells behaved, respectively, in the same way as BALL and TALL cells. Both BALL and TALL cells while directly interacting with the SG cells were completely inhibited from incorporating [3H]thymidine, although radioactive grains were observed in 16.4%-12.4% of supernatant BALL and 13.8%-13.0% of supernatant TALL cells in each coculture. Furthermore the media conditioned by SG cells significantly increased the incorporation of [3H]thymidine into the TALL cells as much as 190% of the control. These results indicate that SG cells undergo tissue-specific cellular interactions with B- and T-lymphoid cell lines but not with a myeloid cell line, and they can modify their growth by two distinct mechanisms. SG cells proved to be very useful in studying the effect of the lymphoid microenvironment on the proliferation of lymphocytes in vivo.

Donor stromal cells support hematopoiesis in CFU-S colonies.

To examine the effect of hematopoietic stromal cells on bone marrow transplantation (BMT), CF-1 cells, which were previously established from splenic stroma of a mouse that was administered recombinant human granulocyte colony-stimulating factor (rhG-CSF), were transplanted with bone marrow cells. The stromal cells were transfected with the plasmid DNA of beta-galactosidase (beta G-CF-1) cells for studying immunohistochemistry. Using immunohistochemistry, the spleen and bone marrow of the recipient mice were examined for beta-galactosidase on the 8th and 12th day after BMT. In the spleen, beta G-CF-1 cells were observed within and in the regions immediately surrounding the colony-forming unit-spleen (CFU-S) on the 8th and 12th day after BMT. In certain regions, beta G-CF-1 cells existed in or surrounded the CFU-S colonies that consisted of the undifferentiated hematopoietic cells, while in other regions the granulopoiesis increased, especially in correspondence with the existence of beta G-CF-1 cells. In the bone marrow cavity, beta G-CF-1 cells were also recognized, though hematopoiesis was lower than the normal bone marrow. These data demonstrate that CF-1 cells, that is, donor stromal cells, can be transplanted to the hematopoietic organs and can then support hematopoiesis.

Passive immunization with anti-parathyroid hormone-related protein monoclonal antibody markedly prolongs survival time of hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors.

Malignancy-associated hypercalcemia is mainly caused by excessive production of parathyroid hormone-related protein (PTHrP) by the tumor. Using anti-PTHrP-(1-34) monoclonal murine antibody (anti-PTHrP MoAb), we studied whether repeated injection of the homologous antibody would continuously decrease the serum calcium concentration in hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors, leading to prolongation of their survival time. Daily SC injections of anti-PTHrP MoAb decreased the serum calcium concentration almost to within the normal range in nude mice bearing transplanted human PTHrP-producing tumors (T3M-1, EC-GI, PC-3, and FA-6) but not in a nude mouse bearing a transplanted parathyroid carcinoma. The antibody did not affect FA-6 tumor growth either in vitro or in vivo. Pancreatic carcinoma cells (FA-6), which caused the most severe hypercalcemia, were inoculated into 6-week-old nude mice. When severe hypercalcemia (approximately 19 mg/dl) had developed, daily SC injection of anti-PTHrP MoAb was started. Within 18 days of this time point, all untreated tumor-bearing mice (n = 10) died of hypercalcemia and cachexia, whereas all the treated mice (n = 10) showed an increase in body weight and survived for at least 25 days. Histologic examination of the treated mice revealed a marked decrease in osteoclastic bone resorption, without toxicologic findings in the kidney and liver. These results suggest that passive immunization against PTHrP can continuously ameliorate the hypercalcemia and markedly prolong the survival time of severely hypercalcemic, tumor-bearing mice. If a human monoclonal antibody against PTHrP-(1-34) could be developed, then passive immunization would be potentially one of the most effective therapies for patients with malignancy-associated hypercalcemia due to excessive production of PTHrP.

Osseous changes and abnormalities of mineral metabolism in rats with glycopeptide-induced nephritis.

A laboratory model of renal osteodystrophy was developed in rats by a single injection of glycopeptide isolated from renal cortical tissues of rats according to the method used by Shibata et al. to induce glomerulonephritis. Approximately 60-70 days after injection, severe proteinuria appeared and continued for at least 170 days at a rate of more than 1 g/day. Morphological changes in the kidney were typical of chronic glomerulonephritis. The plasma calcium concentration was lowered transiently by the 96th day after injection, but was restored to the normal range thereafter. Plasma parathyroid hormone levels, however, continued to rise in parallel with the degree of proteinuria. Marked secondary hyperparathyroidism was induced which led to severe bone atrophy. Histological examinations showed a marked increase of resorbing cavities, with a quantitatively larger number of osteoclasts in cortical bone tissues compared with the control animals. No spontaneous remission was observed. It is emphasized that all of the biochemical and morphological changes reported here were induced by a single injection of homologous renal glycopeptide, and they were highly reproducible.

Comparative therapeutic effects of vitamin D3 and its derivatives on experimental renal osteodystrophy.

The comparative effectiveness of vitamin D3 and its derivatives in curing hyperparathyroidism and osteodystrophic bone lesions was examined in a laboratory model of renal osteodystrophy associated with marked secondary hyperparathyroidism in rats. The experimental model was prepared by a single injection of homologous glycopeptide. Plasma levels of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] appeared to decrease in the rats receiving glycopeptide. Various doses of vitamin D3 derivatives [2 or 10 microgram/kg D3, 2 microgram/kg 25-hydroxyvitamin D3 (25OHD3), 0.1 microgram/kg 1 alpha,25(OH)2D3, and 0.1 or 0.2 microgram/kg 1 alpha-hydroxyvitamin D3 (1 alpha OHD3)] were daily administered orally to the nephritic rats for 23 days before sacrifice. 1 alpha,25(OH)2D3 and 1 alpha OHD3 were much more potent than 25OHD3 and D3 in reducing the hyperplasia of parathyroir glands. The potency of 1 alpha OHD3 in curing the histological changes of osteodystrophy appeared to be greater than that of 1 alpha,25(OH)2D3. The same dose level of 1 alpha OHD3 was more effective than 1 alpha,25(OH)2D3 in enhancing plasma 1 alpha,25(OH)2D3 levels.

Progesterone metabolism in recombinant yeast simultaneously expressing bovine cytochromes P450c17 (CYP17A1) and P450c21 (CYP21B1) and yeast NADPH-P450 oxidoreductase.

Simultaneous expression plasmids were constructed for bovine adrenal cytochromes P450c17 and P450c21 (pA gamma alpha) and for both P450s together with NADPH-cytochrome P450 reductase (pAR gamma alpha). On introduction of each of the plasmids into Saccharomyces cerevisiae AH22 cells, the transformed yeast strains AH22/pA gamma alpha and AH22/pAR gamma alpha produced about 10(5) molecules per cell of P450c17 and 2 x 10(3) molecules per cell of P450c21. The expression levels of NADPH-cytochrome P450 reductase was about 3 x 10(4) and 6 x 10(5) molecules per cell in the strains AH22/pA gamma alpha and AH22/pAR gamma alpha, respectively. When progesterone was added to growing cell cultures of the transformed yeast strains, the substrate was metabolized more rapidly in the AH22/pAR gamma alpha cells than AH22/pA gamma alpha cells, probably due to overproduction of the reductase. In the AH22/pAR gamma alpha cells, progesterone was first converted into 17 alpha-hydroxyprogesterone to the extent of 82% by the catalysis of P450c17. 17 alpha-hydroxyprogesterone was further converted into 11-deoxycortisol by P450c21 to the extent of 60% of the added substrate. The conversion of progesterone into androstenedione through 17 alpha-hydroxyprogesterone was estimated to be less than 3%, suggesting very low C17,20-lyase activity of P450c17, although other hydroxylation products were detected. Androstenedione was further converted into testosterone by an unknown pathway present in S. cerevisiae cells.

Nucleotide sequence of the insecticidal protein gene of Bacillus thuringiensis strain aizawai IPL7 and its high-level expression in Escherichia coli.

A DNA fragment carrying the insecticidal protein gene of Bacillus thuringiensis subsp. aizawai IPL7 was cloned from a 78-kb plasmid. The nucleotide sequence revealed that the cloned DNA fragment contained a 3465-bp protein-coding region with 156-bp 5'-flanking, and 168-bp 3'-flanking regions. The open reading frame encoded a 130,690 Da protein consisting of 1155 amino acid residues. Nucleotide sequence comparison of the aizawai gene with the published berliner 1715 gene showed only 8 nt changes in the coding regions. It was found that 72 bp of the 5'-flanking sequence of the cloned aizawai gene was responsible for constitutive expression of the 130-kDa protein gene in Escherichia coli. The expression was greatly enhanced by introducing the tac promoter upstream from the 72-bp 5'-flanking region of the aizawai gene. Under optimal conditions, the 130-kDa insecticidal protein amounted to 38% of the total cellular protein.

Expression of rat liver vitamin D3 25-hydroxylase cDNA in Saccharomyces cerevisiae.

The cDNA coding for the precursor protein of rat liver mitochondrial vitamin D3 25-hydroxylase, cytochrome P450LMT25, was expressed under the control of the yeast alcohol dehydrogenase I promoter and terminator in Saccharomyces cerevisiae AH22 cells. The transformed yeast cells produced a P450LMT25 protein with an almost similar apparent molecular weight as compared with that of the authentic mature enzyme. The expression level of the P450LMT25 hemoprotein was about 5 x 10(4) molecules per cell as determined by reduced CO-difference spectra. The mitochondrial fraction prepared from the transformed yeast cells exhibited both 25-hydroxylase activity toward 1 alpha-hydroxyvitamin D3 and 27-hydroxylase activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol in a reconstituted system containing bovine adrenodoxin and NADPH-adrenodoxin reductase.

A murine model for bone marrow metastasis established by an i.v. injection of C-1300 neuroblastoma in A/J mice.

A reproducible tumor model for bone marrow metastasis has been developed by an injection of murine C-1300 neuroblastoma (C-1300 NB) cells into the tail vein of syngeneic A/J mice. The animals died with liver metastases at 18-21 days after an injection of 10(5) tumor cells and often had bone marrow metastasis in the femur. N-methylformamide (NMF), a maturational agent, was administered to inhibit liver metastases and to extend survival in mice with advancing bone metastasis. Histological examination of bone marrow metastasis, demonstrated lesions varying from a few small colonies of C-1300 NB cells either in metaphysis or diaphysis to large foci replacing normal hematopoietic bone marrow, simultaneously invading epiphysis or cortex of bone as bone metastasis. This assay demonstrated the ability to detect neuroblastoma cells in the bone marrow histologically and could determine bone marrow TD50 by extraction of bone marrow cells after treatment with various doses of drug. Fifty per cent of mice injected with cyclophosphamide (CY) developed bone marrow metastasis without liver metastasis. Treatment with tamoxifen, an anti-calmodulin drug, suppressed tumor takes in the recipient mice with tamoxifen-dose-dependent fashion. This experimental system allows for investigations into the therapeutic response and biology of neuroblastoma metastases in the bone marrow.

Development of an in situ toxicity assay system using recombinant baculoviruses.

A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is described. Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or more cloned enzymes. The ability of these enzymes to prevent or enhance the toxicity of drugs and xenobiotics is then measured in situ. Initial parameters for the system were developed and optimized using baculoviruses engineered for expression of the mouse soluble epoxide hydrolase (msEH, EC 3.3.2.3) or the rat cytochrome P4501A1. SF-21 cells expressing msEH were resistant to trans-stilbene oxide toxicity as well as several other toxic epoxides including: cis-stilbene oxide, 1,2,7,8-diepoxyoctane, allylbenzene oxide, and estragole oxide. The msEH markedly reduced DNA and protein adduct formation in SF-21 cells exposed to [3H]allylbenzene oxide or [3H]estragole oxide. On the other hand, 9,10-epoxyoctadecanoic acid and methyl 9,10-epoxyoctadecanoate were toxic only to cells expressing sEH, suggesting that the corresponding fatty acid diols were cytotoxic. This was confirmed by showing that chemically synthesized diols of these fatty acid epoxides were toxic to control SF-21 cells at the same concentration as were the epoxides to cells expressing sEH. A recombinant baculovirus containing a chimeric cDNA formed between the rat P4501A1 and the yeast NADPH-P450 reductase was also constructed and expressed in this system. A model compound, naphthalene, was toxic to SF-21 infected with the rat P4501A1/reductase chimeric co-infecting SF-21 cells with either a human or a rat microsomal EH virus along with P4501A1/reductase virus. These results demonstrate the usefulness of this new system for experimentally analyzing the role of enzymes hypothesized to metabolize endogenous and exogenous chemicals of human health concern.

Regional comparison of prolactin gene expression in the human decidualized endometrium in early and term pregnancy.

Prolactin (PRL) is known to be expressed in the decidualized human endometrium and secreted into amniotic fluid. Although the site of synthesis of endometrial PRL is known to be the decidual cells, the difference in PRL gene expression within each area of decidua, i.e. decidua basalis, decidua parietalis and decidua capsularis, during pregnancy is not clear. We have applied an in situ hybridization histochemistry technique using a radiolabeled RNA probe to compare the difference in expression of PRL gene within each area of the decidualized endometrium. Specific hybridization signals were distributed over the decidual cells in early and term pregnancy. More intense hybridization signals were always detected in the tissues of early pregnancy than in those of term pregnancy. In the decidua capsularis of early pregnancy, labeled cells were concentrated close to the amniotic cavity, whereas cells were concentrated close to the maternal surface of the fetal membrane in term pregnancy. In the decidua parietalis, almost all decidual cells were labeled, but no specific labeling was seen in the endometrial glands or capillary endothelium in both groups. In the decidua basalis, most decidual cells showed hybridization signals whereas no hybridization signal was seen over the trophoblast cells. These results show that there are regional and periodic differences in PRL gene expression in the decidual cells during pregnancy.