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H Masui

Publications and source records attributed to H Masui.

At least 73 records · Page 4Linked to original sources
PubMed1990

Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation.

MCF-10A cells are a spontaneously immortalized normal human mammary epithelial cell line. MCF-10A cells were transfected with two expression vector plasmids containing either a human point-mutated c-Ha-ras protooncogene or the rat c-neu protooncogene. c-Ha-ras-transfected MCF-10A cells grow as colonies in soft agar, exhibit a 3- to 4-fold increase in their growth rate in serum-free medium, and show a reduced mitogenic response to exogenous epidermal growth factor (EGF) or transforming growth factor-alpha (TGF alpha) as compared to MCF-10A cells. c-Ha-ras-transfected MCF-10A cells express a 4- to 8-fold increase in TGF alpha mRNA levels and secrete 4- to 6-fold more TGF alpha protein as compared to MCF-10A cells. Addition of either an anti-TGF alpha neutralizing monoclonal antibody or an anti-EGF receptor blocking monoclonal antibody to the Ha-ras-transformed MCF-10A cells produces a 50 to 80% inhibition of colony formation of these cells in soft agar. c-neu-transfected MCF-10A cells grown in soft agar and exhibit an increase in their growth rate in serum-free medium at a level comparable to that observed in Ha-ras-transformed MCF-10A cells. Addition of an anti-c-erbB-2 monoclonal antibody inhibits the anchorage-independent growth of these cells in soft agar. However, c-neu-transformed MCF-10A cells show no increase in TGF alpha secretion and no change in their responsiveness to exogenous EGF or TGF alpha. A recombinant retroviral vector containing the human TGF alpha gene was also introduced into MCF-10A cells. TGF alpha-infected MCF-10A cells secrete 15- to 20-fold more TGF alpha protein than MCF-10A cells, form colonies in soft agar, exhibit an enhanced growth rate in serum-free medium, and show a decreased mitogenic response to exogenous EGF or TGF alpha at a level equivalent to Ha-ras-transformed MCF-10A cells. Growth of TGF alpha-infected MCF-10A cells in soft agar is completely inhibited by anti-TGF alpha neutralizing or anti-EGF receptor blocking monoclonal antibodies. These results suggest that TGF alpha is an intermediary in the transformation of human mammary epithelial cells by an activated c-Ha-ras gene, but not by the c-neu gene, and demonstrate that overexpression of this growth factor is able to transform immortalized human mammary epithelial cells which also express a sufficient complement of functional EGF receptors.

Blotting, Northern↗
PubMed1990

Retinoic acid causes a decline in TGF-alpha expression, cloning efficiency, and tumorigenicity in a human embryonal cancer cell line.

The human teratocarcinoma NTERA-2 cl. D1 (NT2/D1) cell is a cloned multipotential embryonal cancer cell line that differentiates into a neuronal phenotype and other cellular lineages with retinoic acid (RA) treatment. Here we report that mRNA for the transforming growth factor-alpha is expressed in these RA-untreated cells and that RA-treatment results in a reduction of mRNA expression within 24 hr of treatment. In total cellular RNA, TGF-alpha mRNA is not detectable by Northern analysis at 6 days when there is increased expression of the human homeotic genes Hu-1 (Hox 2.1) and Hu-2 (Hox 2.2), known markers of RA response in NT2/D1 cells. RA treatment also causes a marked reduction in cloning efficiency and tumorigenicity of these cells. The addition of TGF-alpha or EGF (epidermal growth factor) protein to RA-untreated NT2/D1 cells augments soft agar cloning under limited fetal calf serum conditions. Blocking monoclonal antibodies directed against the EGF receptor (EGFr) can prevent this augmentation. We conclude that TGF-alpha expression inversely correlates with the state of RA-induced differentiation of this human teratocarcinoma cell and that TGF-alpha and EGF proteins are stimulatory growth factors in NT2/D1 cells under these culture conditions.

Cell Differentiation↗
PubMed1989

Imaging of human tumor xenografts with an indium-111-labeled anti-epidermal growth factor receptor monoclonal antibody.

The mouse monoclonal antibody (mAb) 225 IgG1 against the epidermal growth factor (EGF) receptor has been investigated for its capacity to localize in human tumor xenografts. The EGF receptor is the product of the c-erb-B proto-oncogene (also known as EGFR). Elevated expression of EGF receptors has been demonstrated in many human tumors and tumor cell lines. We studied A431 human vulvar squamous cell carcinoma cells, with 2 X 10(6) receptors per cell; MDA-MB-468 (MDA 468) human breast adenocarcinoma cells, with 3 X 10(5) receptors per cell; and MCF-7 human breast adenocarcinoma cells, with 5 X 10(3) receptors per cell. The 111In-labeled pentetic acid (DTPA), derivative of mAb 225 (111In-DTPA-225) was injected intraperitoneally into nude mice bearing subcutaneous tumor xenografts. We measured uptake by quantifying radioactivity in tumor and normal tissues and by obtaining gamma camera images. Uptake in A431 xenografts was 28% +/- 2.4% of the injected dose per gram of tumor on day 3 and 12.4% +/- 3.0% on day 7. Distribution ratios comparing uptake in the tumor with that in normal tissues were consistently greater than 4. In contrast, there was far less uptake of the control mAb KS1/4S-1 labeled with 111In. This conjugate, 111In-DTPA-KS1/4S-1, has an IgG1 isotype but does not bind to human or murine cells. Imaging of the tumor with mAb 225 was excellent, especially on days 3-7. MDA 468 xenografts exhibited reduced localization of mAb 225 in the tumor. For MCF-7 xenografts, the tumor uptake of mAb 225 after 7 days was only 0.70% +/- 0.10% of the injected dose per gram of tumor, which was comparable to the uptake of the KS1/4S-1 control mAb. The ratio of the concentration of radioactivity in the tumor to that in normal tissue (distribution ratio) showed poor selectivity of uptake, and imaging was not obtained. These observations suggest that labeled mAb can target the product of a proto-oncogene, the EGF receptor, when it is expressed at high levels in human tumor xenografts.

Animals↗
PubMed1989

Cytotoxicity against human tumor cells mediated by the conjugate of anti-epidermal growth factor receptor monoclonal antibody to recombinant ricin A chain.

We have produced monoclonal antibodies against the epidermal growth factor (EGF) receptor which bind to the receptor with high affinity, compete with EGF for binding, block EGF-induced tyrosine kinase activity, and activate internalization and down-regulation of the receptor. These antibodies are cytostatic against cultured A431 cells at concentrations of 5-20 nM. In addition, they prevent the growth of A431 tumor xenografts in athymic mice. In the present experiments, we have attempted to improve the antitumor activity of monoclonal antibody 528 IgG2a against the EGF receptor by linking it to recombinant ricin A chain (rRA). The immunoconjugate (528 IgG-rRA) showed a potent cytotoxic effect on A431 cells in vitro. At a concentration of 10 pM, it inhibited the proliferation of cultured A431 cells by 50% and also inhibited protein synthesis in these cells by 50%. Proliferation was prevented and cell death occurred at 528 IgG-rRA concentrations of 60 pM or greater. Recombinant free ricin A chain was far less toxic. The cytotoxic effect of the immunoconjugate was neutralized by 528 IgG at concentrations 100-fold higher than 528 IgG-rRA. When the cytotoxic effect of 528 IgG-rRA was compared among several human cell lines expressing different numbers of EGF receptors, the capacity to inhibit both proliferation and protein synthesis generally correlated with the number of EGF receptors on the plasma membranes of these cells. Since 528 IgG-rRA is a very potent immunotoxin against A431 cells in culture, we designed experiments to test its in vivo antitumor activity against A431 xenografts in athymic mice. To measure the clearance of 528 IgG-rRA, 50 micrograms of immunotoxin were injected i.p. into athymic mice, blood was collected from the animals at regular intervals, and the level of immunotoxin in the serum was assayed by protein synthesis inhibition in cultured A431 cells. The blood level of active immunoconjugate reached a maximum 6 h after i.p. injection. The half-life of the absorption phase was 2.2 h, the half-life for elimination was 9.2 h, and blood levels which could be potentially cytotoxic were maintained for 48-72 h. We investigated a number of immunotoxin treatment schedules, including every other day for 4 days, based on these data. The results demonstrate that, while 528 IgG-rRA has higher in vivo antitumor activity than 528 IgG against A431 cell xenografts, this is accompanied by toxicity against the murine host.

Animals↗
PubMed1989

Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells.

The response of malignant and nonmalignant human breast cell lines to the growth inhibitory effects of monoclonal antibodies against the epidermal growth factor (EGF) receptor was studied. A series of human breast cell lines, which express EGF receptor, were used: MDA-468, MDA-231, and Hs578T human breast cancer cells and the transformed human mammary epithelial cell lines 184A1N4 and 184A1N4-T that have been benzo[a]pyrene immortalized and further transformed with SV40T, respectively. Four antibodies of two different classes were tested: 225 immunoglobulin G (IgG), 108.4 IgG, 96 immunoglobulin M (IgM), and 42 IgM. All four antibodies inhibited the anchorage-dependent and -independent, EGF-stimulated growth of 184A1N4 and 184A1N4-T cells, respectively, and this growth inhibition could be reversed by the addition of increasing concentrations of EGF. In contrast, the antibodies inhibited the anchorage-dependent and -independent growth of MDA-468 cells in the absence of exogenous EGF suggesting that the antibodies were acting to block access of an endogenously produced ligand to the EGF receptor. In the presence of antibody and increasing concentrations of EGF, MDA-468 cell growth was first stimulated then inhibited as the EGF concentration increased, thus, uncovering the growth stimulatory potential of low concentrations of EGF in these cells. Data is presented that indicates MDA-468 cells secrete a transforming growth factor with autocrine growth stimulatory capabilities. The growth of MDA-231 and Hs578T cells, which contain activated ras oncogenes, was not inhibited by the antibodies and the growth of these cell lines was not stimulated by EGF. Of the cell lines studied only MDA-468 cells appear to possess an autocrine growth stimulatory capacity.

Antibodies, Monoclonal↗
PubMed1989

Preparation and release characteristics of cisplatin albumin microspheres containing chitin and treated with chitosan.

Cisplatin (CDDP) containing albumin microspheres and microcapsules incorporating biodegradable macromolecules, chitin and chitosan, were prepared, and their CDDP content and releasing ability and susceptibility to various enzymes were examined. Chitin was incorporated during preparation of the microspheres, while chitosan was used to treat preformed microspheres. CDDP content was remarkably increased by chitin; when chitin was incorporated at a concentration of 1.5%, the CDDP content of the microspheres was found to be 16.2% (1.8 times that with no addition of chitin). CDDP release was suppressed by chitin and chitosan. The 50% CDDP release time was about 1.5 h when no chitin was added, but about 16 h was required when chitin was incorporated into the microspheres at a concentration of 1.5%. Chitin and chitosan suppressed the decomposition by protease. The microspheres treated with 70% deacetylated chitosan showed the greatest susceptibility to lysozyme. In conclusion, CDDP release can be controlled by the use of chitin or chitosan, and the microspheres should show no immunogenicity in vivo because of their susceptibility to lysozyme.

Albumins↗
PubMed1987

Evidence that neurokinin A (substance K) neurons project from the striatum to the substantia nigra in rats.

The striatonigral neurokinin A (NKA) pathway in rats was examined with an antiserum specific to the N-terminal region of NKA. Thermal lesions of the striatum decreased both substance P-like immunoreactivity (SP-LI) and NKA-like immunoreactivity (NKA-LI) in the substantia nigra (SN). The releases of SP-LI and NKA-LI in the SN were measured by a push pull superfusion technique. Addition of 50 mM K+ to the superfusion medium increased the releases of both SP-LI and NKA-LI to 1.5-2.0 times the basal values. These results suggest that NKA neurons as well as SP neurons in the striatum project to the SN in rats.

Animals↗
PubMed1987

Activity of cancer chemotherapeutic agents against human colorectal carcinomas grown as primary tissue culture.

Improved procedures are described for the seeding of primary cultures from human colon adenocarcinoma and for the use of these cultures in the evaluation of drug effects. Two of the specimens studied were xenografts maintained in athymic (nude) mice, while the other six were biopsies obtained directly from patients. Tumor cells obtained directly from the patients proliferated in defined hormone-supplemented medium to the exclusion of other cells. In drug-response studies with cultures from a colon tumor biopsy all four drugs studied (4'-deoxydoxorubicin, 4'-O-methyldoxorubicin, 5-fluorouracil, and 1,3-bis-[chloroethyl]-1-nitrosourea) inhibited growth of the cells within 3-6 days after drug treatment. On an equitoxic dose basis (LD10 in mice), 4'-deoxydoxorubicin appeared to be the most active drug. This drug also showed dose-dependent activity against one of the xenografted tumors in vitro. In dose-response studies with cultures from another patient's colon tumor, doxorubicin and 5-fluorouracil showed significant activity against the tumor 10 days after the drug treatment with concentrations at 1X and 10X average peak plasma levels.

Animals↗
PubMed1987

A functional role of cholinergic innervation to neurons in the cat visual cortex.

1. Effects of microionophoretic application of acetylcholine (ACh) and its antagonists on neuronal responses to visual stimuli and to electrical stimulation of the lateral geniculate nucleus were studied in the cat striate cortex. 2. Responses elicited visually and electrically were facilitated by ACh in 74% of the cells tested, whereas the responses were suppressed in 16%. These ACh effects were blocked by a muscarinic antagonist, atropine, but not by a nicotinic antagonist, hexamethonium, indicating that the ACh effects are mediated through muscarinic receptors. A single application of atropine suppressed visual responses of cells facilitated by ACh, whereas it enhanced those of cells inhibited by ACh, suggesting that endogenous ACh may tonically modulate visual responsivity of cortical neurons. 3. In most cells with the facilitatory ACh effect, responses with single spikes to the electrical stimulation became more consistent, often with double spikes, during the ACh application. The suppressive effects of ACh were noted most often in cells with a longer response latency to electrical stimulation of lateral geniculate nucleus. 4. In most of the facilitated cells the spontaneous activity remained null or very low during ACh application, in spite of marked enhancement of visual responses, suggesting that ACh may improve the signal-to-noise ratio (S/N) of cortical neuron activity. To confirm this suggestion, we calculated a S/S + N index by counting the total number of spikes in the responses (S) and that in peristimulus time histogram (S + N) and found that it was improved during the ACh application in about a half of the cells, whereas it became worse in about one-fifth. 5. In most of the facilitated cells, ACh enhanced visual responses not only to optimal but also to nonoptimal stimuli, resulting in no improvement or even worsening of the orientation selectivity. This was also the case in the selectivity of direction of stimulus movement. 6. The laminar location of the facilitated cells was biased toward layers V and VI of the cortex, although they also made up the majority in layers II + III and about half the tested cells in layers IVab and IVc. 7. In the light of recent understanding of cortical circuitry, these results suggest that the cholinergic innervation to cortical neurons may play a role in improvement of the S/N ratio of information processing in the striate cortex and in facilitation of sending processed informations to other visual centers.

Acetylcholine↗
PubMed1987

Epidermal growth factor receptor expression in human lung carcinomas defined by a monoclonal antibody.

Acetone-fixed frozen tissue sections from 56 cases of human lung carcinoma were tested for reactivity by an indirect immunoperoxidase technique with a monoclonal antibody (MAb 528) specific for the external domain of the epidermal growth factor receptor (EGFR). MAb 528 reacted with all epidermoid (22/22) and large-cell (4/4) lung carcinomas evaluated. The antibody was also positive with a subset of lung adenocarcinomas (13/21) and did not react with small-cell lung cancers (SCLCs) (0/9). MAb 528 also stained normal bronchial epithelium identified within the tumor sections of 5 cases. Thus EGFR was expressed by all epidermoid and large-cell lung carcinomas examined, a subset of lung adenocarcinomas, and normal bronchial epithelium. EGFR expression was not identified in any of the SCLCs tested. These data imply that immunohistochemical detection of EGFR expression may find future application in distinguishing epidermoid, large-cell, and some adenocarcinomas of the lung from SCLCs.

Adenocarcinoma↗
PubMed1987

Anti-epidermal growth factor receptor monoclonal antibodies may inhibit A431 tumor cell proliferation by blocking an autocrine pathway.

Monoclonal antibodies which bind to the EGF receptor have the capacity to inhibit EGF-induced effects upon proliferation and biochemical functions in cultured human cells. Some EGF receptor-bearing tumor cells are prevented from growth by treatment with antireceptor antibody. Evidence is presented which suggests that antibody-mediated antiproliferative activity may result from effects upon growth factor-dependent processes in the receptor-bearing cells.

Antibodies, Monoclonal↗
PubMed1986

Distribution of substance K (neurokinin A) in the brain and peripheral tissues of rats.

The regional distribution of substance P (SP) and substance K (SK) in the central nervous system and peripheral tissues of rats was studied. High levels of SK-like immunoreactivity (SK-LI) and of SP-like immunoreactivity (SP-LI) were found in the substantia nigra, nucleus tractus solitarius and nucleus habenula. Neonatal treatment of rats with capsaicin caused a significant decrease in the SK-LI content in the submaxillary gland, but capsaicin had no effect on SK-LI content in any area of the brain. These observations suggest that SK in the brain and peripheral tissues may have significant physiological roles.

Animals↗
PubMed1986

Excitatory amino acid transmitters in neuronal circuits of the cat visual cortex.

To test a possibility that glutamate (Glu) and aspartate (Asp) are transmitters in the visual cortex and to locate their operating sites in the cortical circuitry, we studied effects of microiontophoretic application of Glu/Asp antagonists on visual responses of cortical neurons in the cat. The antagonists tested were kynurenic acid (KYNA), cis-2,3-piperidine dicarboxylic acid, and gamma-D-glutamylglycine. Among these antagonists, KYNA was most effective in blocking visual responses of cortical neurons; it eliminated visual responses in 156 of the 188 cells tested. Usually the maximal suppressive effect appeared 20-30 s after starting KYNA application and recovery of cell's responsiveness 30-60 s after stopping the application. KYNA antagonized excitations induced by ionophoretic application of Glu and Asp but did not block those by acetylcholine, suggesting that KYNA is a selective antagonist of Glu/Asp, and its action is not due to general depressant effects. This suggestion was further supported by the observation that in corticogeniculate cells the latency and probability of invasion of antidromic spikes into the somatodendritic part following electrical stimulation of the lateral geniculate were not changed while visual responses were completely suppressed by KYNA. In terms of actions of the three agonists which give the basis for classifying excitatory amino acid receptors into at least three types, KYNA antagonized excitations by N-methyl-D-aspartic acid (NMDA) and kainate in almost all the cells tested but did not block those by quisqualate in about half of the cells. These results suggest that KYNA reacts more preferentially with NMDA and kainate receptors than with quisqualate receptors. Effectiveness of KYNA was related to types of receptive fields of cells and to their laminar locations. In 79 of the 104 simple cells tested, KYNA completely suppressed their visual responses, while such a complete block was seen in only 18 of the 68 complex and 3 of the 16 special complex cells. The great majority of the cells in layers IVab, IVc and the upper part of layer VI were completely suppressed by KYNA, whereas most of the cells in the other layers were incompletely suppressed or not suppressed at all.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1986

Effects of epidermal growth factor receptor concentration on tumorigenicity of A431 cells in nude mice.

To test the relationship between the concentration of epidermal growth factor (EGF) receptors and tumor growth in vivo, we measured the rate of growth of several independently isolated A431 cell lines in athymic mice. This series of A431 clonal variants with differing extents of EGF receptor gene amplification and protein expression were implanted into athymic mice and the time to solid tumor formation and rate of growth were measured. Results of these experiments indicate that the degree of gene amplification and concentration of EGF receptors are directly correlated with the growth of these cells as solid tumors in host animals. Complementary DNA hybridization analysis revealed no change in the extent of gene amplification and expression in implanted cells versus excised tumors nor any evidence of further gene rearrangement in vivo. A high concentration of EGF receptors appears to facilitate the growth of tumor cells in vivo and in vitro.

Animals↗
PubMed1986

A novel monoclonal antibody-defined antigen which distinguishes human non-small cell from small cell lung carcinomas.

Spleen cells from BALB/c mice hyperimmunized with the human epidermoid lung carcinoma cell line T222 were fused with NS-1 mouse myeloma cells to produce monoclonal antibodies to human lung cancer antigens. Hybridoma culture supernatants were tested by an enzyme-linked immunosorbent assay for reactivity against a panel of human lung tumor cell lines. Supernatant from hybridoma EA1 (immunoglobulin G1) displayed strong reactivity with four of four non-small cell lung carcinomas but did not react with three of three small cell lung carcinoma (SCLC) cell lines. This hybridoma was cloned by limiting dilution and utilized to generate ascites antibody for subsequent immunohistochemical and antigen characterization studies. Evaluation of fresh frozen tumor tissue sections by immunoperoxidase staining methods revealed EA1 reactivity with the vast majority of non-SCLCs tested (21 of 21 epidermoid, 17 of 18 adenocarcinomas, four of four large cell, two of two bronchioloalveolar) and no reactivity with nine of nine small cell lung carcinomas. EA1 also stained bronchial epithelium and other benign and malignant epithelial tissues. The EA1 antigen was determined to have a molecular weight of 75,000 by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of human non-SCLC tumor extracts. These data imply that EA1 recognizes a novel antigen expressed by non-SCLCs and other epithelial tissues. The absence of EA1 reactivity with SCLCs suggests that this monoclonal antibody may find future application in distinguishing non-SCLC from SCLC and prove useful in furthering our understanding of the histogenesis of lung carcinomas.

Adenocarcinoma↗
PubMed1986

Mechanism of antitumor activity in mice for anti-epidermal growth factor receptor monoclonal antibodies with different isotypes.

In previous studies of monoclonal antibodies (mAbs) against the receptor for epidermal growth factor (EGF) both 528 IgG2a and 225 IgG1 were shown to inhibit growth of A431 cell xenografts in athymic mice. The antitumor activities of the two mAbs were similar and, although they differ in their isotypes, they share many properties. The two mAbs bind to EGF receptors with identical affinities, compete with EGF for binding to EGF receptors, down regulate the receptors identically, block EGF-induced activation of tyrosine protein kinase activity to a comparable degree, and block EGF-induced changes in the proliferation of cultured cells. These similarities in physiological effects permit a direct comparison of the mechanisms of action mAbs of the IgG1 and IgG2a isotypes. We examined in vitro cytotoxicity against A431 cells, using 528 IgG2a and 225 IgG1 mAbs. 528 IgG2a, but not 225 IgG1, demonstrated partial complement-mediated cytotoxicity by the 51Cr release assay and by growth inhibition of cultured A431 cells. 528 IgG2a, but not 225 IgG1, was cytotoxic to A431 cells in the presence of activated peritoneal macrophages, as demonstrated by release of incorporated [3H]thymidine. Neither mAb showed any significant cytotoxicity to A431 cells in the presence of nonadherent spleen cells which contain K-cells. The results of in vitro cytotoxicity experiments suggested that the antitumor activity of 528 IgG2a, but not 225 IgG1, could be mediated by macrophages. This was verified by in vivo experiments in which s.c. tumor cell inocula containing activated macrophages showed enhancement of antitumor effects in animals treated i.p. twice weekly for 3 weeks with suboptimal doses of 528 IgG2a. This enhancement was not observed when 225 IgG1 was used with the same procedure. The results of these experiments suggest that immune mechanisms involving activated macrophages or complement could contribute to the antitumor activity of anti-EGF receptor mAb with the IgG2a isotype, but not with the IgG1 isotype. This observation confirms the findings of others who examined the antitumor activity of IgG2a mAbs in other model systems. IgG1 mAb 225, and possibly IgG2a mAb 528, may prevent growth of human tumor xenografts by altering the physiological functions of the EGF receptor rather than by immune mechanisms.

Animals↗