Comparative studies on estrogen receptors between a pregnancy-dependent mouse mammary tumor (TPDMT-4) and related autonomous tumors.

TPDMT-4 is a pregnancy-dependent mouse mammary tumor line characterized by progressive growth in pregnant, but dormancy in virgin, hosts and by significant estrogen (ER) and progesterone receptor (PR) levels. Three sublines, T4-OR26, T4-0I96, and T4-0I320, were established from sporadic outgrowths in virgins of TPDMT-4 pieces passaged long in breeding and estradiol (E2)- plus progesterone-treated mice and of enzymatically dissociated TPDMT-4 cells, respectively. T4-OR26 tumors produced ovarian-responsive growth and maintained the parent levels of ER and E2-dependent PR. T4-OI320 and T4-OI96 tumors were ovarian independent or autonomous. The former had ER, but not E2-dependent PR, and the latter had neither. To clarify the mechanism of acquisition of autonomy by hormone-dependent neoplastic cells, comparative ER studies were conducted between TPDMT-4 and T4-OI320 tumors. Although cytosolic, microsomal, and nuclear translocated ER levels were generally lower in T4-OI320 tumors, the dissociation constants, determined by the dextran-charcoal technique, steroid specificity, sedimentation data in sucrose gradients, and activation studies using DNA binding, molecular transformation from 4S to 5S, and [3H]E2 dissociation from [3H] E2-ER complexes as markers, revealed no autonomy-specific changes in cytosolic ER. Small but significant differences in the affinity of [3H]E2 to nuclear ER and the [3H]E2 dissociation rate from nuclear bound [3H]E2-ER complexes were found between both tumors. Hormone-dependent tumors may progress toward autonomy with different receptor statuses in different environments, and lack of E2-dependent PR synthesis in ER-positive tumors may be due to defects at postreceptor or nuclear levels involving subtle changes in interaction between activated ER and nuclei.

Analysis of splenic lymphocytes with high electrophoretic mobility in adult and aged nude mice.

Electrophoretic mobility (EPM) and surface markers of splenic lymphocytes in adult (8 weeks old) and aged (over 1 year old) nude mice were investigated. Splenic lymphocytes in nude mice showed a bimodal pattern consisting of low mobility lymphocytes (LML) corresponding to B cells and high mobility lymphocytes (HML). The HML of nude mice showed the following immunological characteristics: (1) surface Ig- cells; (2) asialo GM1+ cells; (3) an increase in natural killer (NK) activity after depletion of B cells; (4) abrogation of the HML peak and NK activity after treatment with anti-asialo GM1 and complement. These findings suggested that HML in nude mice were NK cells. The mobility of NK cells was slightly lower than that of T cells in normal mice, although their histograms greatly overlapped each other. In the spleen cells of nude mice, there was a significant increase in the numbers of Thy-1+ cells and a decrease in the intensity of asialo GM1 antigen as a function of age. The surface markers of HML were Thy-1+- asialo GM1++ in adult nude mice, but were Thy-1+ asialo GM1+ in aged nude mice. However, although HML in aged nude mice became Thy-1+, these had almost the same EPM as those in adult nude mice.

A novel angiogenic inhibitor derived from Japanese shark cartilage (I). Extraction and estimation of inhibitory activities toward tumor and embryonic angiogenesis.

Guanidine extraction and crude fractionation of Japanese shark cartilage by ultrafiltration on a molecular weight basis were conducted and the antiangiogenic activities were assayed as to the inhibitions of tumor and embryonic angiogenesis. Significant inhibition of angiogenesis was found, and there was a linear relationship between the results of the two assays. The inhibitory activities were concentrated in the fraction in the molecular weight range of 103 to 104, and were resistant to heat treatment.

Angiogenic activity of rat mammary carcinomas induced by 7,12-dimethylbenz[a]anthracene and its inhibition by medroxyprogesterone acetate: possible involvement of antiangiogenic action of medroxyprogesterone acetate in its tumor growth inhibition.

The significant inhibitory activity of medroxyprogesterone acetate (MPA) against mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) was confirmed in female Sprague-Dawley (SD) rats, and was found to be independent of the estrogen receptor (ER) level. To facilitate elucidation of the mechanism underlying the antitumor activity of MPA against the rat mammary tumors (RMTs) regardless of ER status, the present study was conducted to determine whether or not a DMBA-induced RMT had the capacity to elicit angiogenic activity on tissue implantation into a rabbit cornea, and, if so, to determine whether or not the angiogenic activity of the RMT was inhibited by MPA. The RMTs obtained were classified into two groups based on the ER level; ER-positive and -negative groups. Both groups exhibited relatively strong angiogenic activity, the activity of the ER-negative group being significantly higher than that of the ER-positive one. The angiogenesis produced by both groups of RMTs was significantly inhibited by MPA, as judged from the results of the rabbit cornea assay. Similarly, MPA almost entirely suppressed not only the angiogenesis but also the growth of rabbit VX2 tumors without ER, included as a positive control as to the induction of angiogenic activity. In vitro experiments using neoplastic epithelioid RMT-1 and -2 cells cloned from DMBA-induced RMTs demonstrated that MPA had little or no suppressive effect on the growth of these epithelial cells. These results suggest that the inhibitory action of MPA toward the angiogenic activity of RMTs, at least in part, involves its antitumor activity toward the RMTs.

A highly potent antiangiogenic activity of retinoids.

Four retinoids, i.e. retinol (vitamin A), retinoic acid, retinyl acetate and synthetic chalcone carboxylic acid (Ch 55), were examined for their effects on embryonic angiogenesis using 4.5-day chorioallantoic membranes of chick embryo. The effects of these retinoids were compared with that of antibiotic herbimycin A, which was the most powerful inhibitor of the angiogenesis reported previously. The four retinoids strongly inhibited embryonic angiogenesis; the order of inhibitory activity was Ch 55 greater than retinoic acid greater than herbimycin A greater than retinyl acetate based on the dose required for the half-maximal inhibitory effect. The present results suggest that retinoids are effective inhibitors of angiogenesis, and can be applied for the management of certain diseases accompanied by aberrant angiogenesis, particularly that which occurs during progressive growth of solid tumors.

Angiogenic factor of a rat mammary tumor cell line (RMT-1) (I). Secretion of two distinct angiogenic factors into serum-free conditioned medium by RMT-1 cells.

Serum-free conditioned medium of a rat mammary tumor cell line RMT-1, established from a rat mammary carcinoma induced by 7,12-dimethylbenz[a]anthracene (DMBA), produced the complete angiogenic response in both rabbit cornea and chick embryo chorioallantoic membrane assays. The angiogenic activity in the RMT-1 conditioned medium was separated into two fractions on a column of heparin-Sepharose; one was eluted with 0.1 M NaCl and the other with 0.5 M NaCl, which are referred to hereafter as rAF-1 and rAF-2, respectively. These two angiogenic factors were further purified separately by FPLC on a Superose 12 column. The partially purified rAF-2 had an apparent Mr of 30,000-50,000 and seemed to exhibit mitogenic activity toward Balb/c 3T3 cells, while the partially purified rAF-1, with an apparent Mr of 10,000-30,000 did not have a mitogenic effect on these cells. Both rAF-1 and rAF-2 were resistant to heat and acid treatment, and exhibited trypsin sensitivity, suggesting that they are heat and acid stable peptides. The two angiogenic factors did not stimulate the proliferation of cultured vascular endothelial cells. These results suggest that RMT-1 secretes two distinct angiogenic factors into the medium and that these two secretable angiogenic factors participate cooperatively in the induction of the angiogenic response produced by a DMBA-induced rat mammary tumor in vivo.

Enkephalin-degrading aminopeptidase in the longitudinal muscle layer of guinea pig small intestine: its properties and action on neuropeptides.

A membrane-bound enkephalin-degrading aminopeptidase was purified from the longitudinal muscle layer of the guinea pig small intestine by four steps of column chromatography using L-tyrosine beta-naphthylamide. The molecular weight of the enzyme was estimated to be 105,000 by gel filtration. The maximum activity was observed between pH 6.5 and 7.0. The Km value for leucine-enkephalin was 137 microM. The aminopeptidase activity toward aminoacyl beta-naphthylamide substrates was restricted to basic, neutral, and aromatic aminoacyl derivatives. No action was detected on acidic amino acid and proline derivatives. The enzyme was potently inhibited by the aminopeptidase inhibitors actinonin, amastatin, and bestatin, and bioactive peptides such as angiotensin III, substance P, and Met-Lys-bradykinin. The enzyme activity was also inhibited by the antibody against the purified serum enkephalin-degrading aminopeptidase of guinea pig at concentrations similar to those at which activity was observed toward serum enkephalin-degrading aminopeptidase and renal aminopeptidase M. The enzyme rapidly hydrolyzed Leu-enkephalin and Met-enkephalin with the sequential removal of the N-terminal amino acid residues. The enzyme also hydrolyzed two enkephalin derivatives, angiotensin III and neurokinin A. However, neurotensin, substance P, and bradykinin were not cleaved. These properties indicated that the membrane-bound enkephalin-degrading aminopeptidase in the longitudinal muscle layer of the small intestine is similar to the serum enkephalin-degrading aminopeptidase and resembles aminopeptidase M. It is therefore suggested to play an important role in the metabolism of some bioactive peptides including enkephalin in peripheral nervous systems in vivo.

Mechanism of the antitumor activity of 5,5'-bis(2'-tetrahydropyranyl) secalonic acid D against Meth-A.

The mechanism of the antitumor activity of 5,5'-bis(2'-tetrahydropyranyl) secalonic acid D (PSA) was examined in Balb/c mice bearing Meth-A fibrosarcoma. IP-injected PSA showed remarkable antitumor activity against IP-implanted Meth-A tumor. Antitumor activity of PSA was not abolished by treatment with silica as an antimacrophage agent or anti-asialo GM1 antiserum that selectively eliminates natural killer cells. Although it was significantly suppressed by treatment with antithymocyte globulin in Balb/c mice, PSA was effective against Meth-A tumors implanted in athymic Balb/c mice. PSA inhibited in vitro Meth-A proliferation as effectively as mitomycin C and was not effective against Meth-A tumor implanted SC at a site where direct contact of PSA and Meth-A cells was unlikely. These results suggest that the antitumor activity of PSA was mainly achieved by inhibiting Meth-A cell proliferation, although the host T cell-mediated immunity was partly involved in the eventual therapeutic efficacy of PSA.

A new aminopeptidase in monkey cerebral membrane fraction: hydrolysis of enkephalin.

A new aminopeptidase, which cleaves the Tyr1-Gly2 bond of enkephalin, was partially purified from the monkey brain membrane fraction. The molecular weight of the enzyme was estimated to be about 53,000, and the optimum pH was in the neutral region (pH 6.5). The enzyme hydrolyzed Leu-enkephalin with a Km value of 238 microM. It strongly hydrolyzed L-tyrosine and L-leucine beta-naphthylamide, but showed only weak affinity for L-arginine or L-alanine beta-naphthylamide. The enzyme was much more potently inhibited by bestatin (IC50: 2 x 10(-8) M) than the other specific aminopeptidase inhibitors examined, while it showed low sensitivity to puromycin and actinonin, inhibitors of cerebral enkephalin-degrading aminopeptidase and aminopeptidase M, respectively. These results indicate that the new enkephalin-degrading aminopeptidase is clearly distinct from aminopeptidase M, which has been reported to be a key enzyme in enkephalin inactivation.

Inhibition of angiogenesis by vitamin D3 analogues.

The effects of vitamin D3 and two analogues on embryonic angiogenesis were studied in 4.5-day-old chick embryo chorioallantoic membranes. The active metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3, and a synthetic vitamin D3 analogue, 22-oxa-1 alpha,25-dihydroxyvitamin D3, inhibited angiogenesis in a dose-dependent manner, the inhibition occurring in the picomolar range. In contrast, vitamin D3 was not effective. The results suggest that these two vitamin D3 analogues might be promising anti-angiogenic agents for controlling the angiogenesis which occurs in several pathological conditions, including tumor development.

A reverse effect of in vitro and in vivo concanavalin A-induced spleen cells on anti-sheep red blood cells response detected with a new method based on flow cytometry.

A simple flow cytometric method for detecting humoral immunity against sheep red blood cells (SRBC) is described. The SRBC were incubated with the serum from SRBC-immunized mice, monoclonal anti-SRBC, or the supernatant which was obtained from the in vitro primary culture of spleen cells with SRBC. The antibodies which bound to SRBC were estimated by means of an immunofluorescence and a flow cytometry. When the channel number of the peak in the histogram of flow cytometry was measured as an index of fluorescence intensity of SRBC, the number significantly correlated with the concentration of IgM and IgG classes of anti-SRBC. The flow cytometry method and hemagglutination (HA) test, as a classic method, were compared in SRBC-immune sera and monoclonal anti-SRBC antibody. The sensitivity determined with flow cytometry was much higher than that with HA. The minimum detectable concentration of anti-SRBC antibody was found to be 3.4 ng/ml by the flow cytometry. The dose response of SRBC in in vitro primary culture was detected by the flow cytometry, not by HA, and the response increased with the dose of SRBC. Using this method, the effect of in vitro and in vivo concanavalin A (Con A)-induced spleen cells on humoral response against SRBC was examined in an in vitro culture system. Anti-SRBC response (IgM and IgG) was found to be suppressed by in vitro Con A-induced lymphocytes but enhanced by in vivo Con A-induced lymphocytes. Thus, this new approach is found to be a good method for detecting the in vitro primary humoral antibody response, which is known to have a low reactivity.

Analysis of electrophoretic mobility histograms of mouse splenocytes and thymocytes during tumor growth and after combined chemotherapy and immunotherapy.

Changes in electrophoretic mobility histograms of splenocytes and thymocytes were studied in plasmacytoma X5563-bearing mice as an indicator of response to treatment with mitomycin C (MMC) alone or combined with the immunomodulator Krestin (PSK). Tumor growth was inhibited by 80-90% in the MMC-treated and was further inhibited in the MMC and PSK-treated group. Electrophoretic mobility histograms of splenocytes were used to determine the fraction of cells having intermediate mobility between high mobility (T cells) and low mobility (B cells). This fraction of intermediate-mobility cells increased in tumor-bearing mice, but a normal electrophoretic mobility pattern was obtained following successful antitumor treatment. In the electrophoretic mobility histogram of thymocytes, on the other hand, the low-mobility cells (cortical thymocytes) decreased in number during tumor growth and were further reduced in the MMC-treated group. This reduction was less in the MMC and PSK-treated group. These results suggest that combined therapy with MMC and PSK prevents damage of the host defence mechanism and allows more efficient antitumor treatment. Analysis of electrophoretic mobility histograms of splenocytes and thymocytes using a fully automated cell electrophoretic instrument makes possible the rapid evaluation of the immunological effects of drug therapy of tumor-bearing mice.

Determination of an immunosuppressive substance in serum by latex particle electrophoresis.

The level of immunosuppressive substance (IS), which increases in the serum of patients with cancer, was determined by an assay based on particle electrophoresis. Polystyrene latex particles (PLP) were coated with IS, which was extracted from the ascitic fluid of patients with cancer. The IS was a glycoprotein with a molecular weight of about 52,000, and an isoelectric point in the range pH 2.7-3.3. When the IS on the surface of the PLP reacted with the anti-IS antibody, the mean electrophoretic mobility of the PLP changed from -3.16 to +0.21 micron.s-1.V-1.cm in the medium of pH 7.2 and ionic strength I = 0.0154. After preincubation of anti-IS antiserum and tested serum, the PLP coated with IS were added to this solution. It was incubated again and then the surface charge of the PLP was measured by an automatic cell-electrophoretic instrument. This method was used to determine the IS concentration in the serum of cancer patients and pregnant women. When compared to healthy controls, the serum IS level was significantly higher in patients with cancer, and lower in pregnant women. The assay based on latex-particle electrophoresis proved to be a sensitive and rapid method for determining the IS level in serum.

[Emergenec of low-mobility lymphocytes in the tumor-bearing status and its immunological significance].

The immunological significance of the lymphocyte mobility histogram derived from a fully automated cell electrophoretic instrument (Parmoquant-L), was considered. In mouse lymphocytes, the ratio of medullary thymocytes increased as a result of a decrease in the proportion of cortical thymocytes, whereas the ratio of splenocytes in the low-mobility zone (0.85-0.95 micron/sec/V/cm) increased in the course of tumor growth or in vivo concanavalin A-treatment. In the peripheral lymphocytes of cancer patients, the decreased ratio of high-mobility T cells resulted in a gradual increase in that of low-mobility T cells, which were not seen in lymphocytes modified by the patient's serum. Although the formation of blastoid cells by stimulation of the tumor or mitogen cannot be completely denied, the low-mobility T cells of peripheral lymphoid tissues in the tumor-bearing status are considered to be caused by incomplete differentiation of the cells, in which immunosuppressor cells are involved. Pattern analysis of lymphocyte electrophoresis would be a simple method if fully automated instrumentation was used.

[Angiogenesis and its regulation].

Angiogenesis (formation of new blood vessels) is essential in embryonal development, female reproduction and wound repair. Under these physiological conditions, angiogenesis is highly regulated by the endogenous angiogenic factor and its inhibitor. However, angiogenic diseases such as rheumatoid arthritis, diabetic retinopathy, solid tumor, hemangioma and psoriasis are driven by persistent unregulated angiogenesis. The angiogenic process consists of such multi-steps as degradation of basement membrane, cell migration and proliferation, and tube formation following activation of vascular endothelial cells by stimulators appearing at the pathological site. Thus, for modulating the pathological conditions, it is most important to elucidate the angiogenic mechanism at the molecular level. It is not sufficiently clear yet as compared with the blood coagulation and hematopoietic systems. On the other hand, there have been advances in the study of new anticancer metastatic agents as a target of activated endothelial cells and they have been found to have promising substances. In this study, the angiogenic factor, its inhibitor, and then the anticancer effect of the angiogenic inhibitor, have been investigated from the standpoint of cancer therapy.

[Analysis of electrophoretic mobility pattern of immunocompetent cells in the tumor-bearing host].

Effect of tumor burden on the electrophoresis of lymphocytes and macrophages was analyzed with a fully automated cell electrophoretic instrument (Parmoquant). The histogram patterns of peripheral lymphocytes of mice shifted to the lower mobility zone, and that of thymocytes shifted reversely, in the course of development of tumor. In cancer patients, emergence of low mobility T cell (LMT) was observed, and LMT correlated with immunosuppressive factors significantly. The ratio of LMT to high mobility T cells (HMT) tended to increase with tumor growth, and showed extremely high value in the case of recurrent cancers. In the follow-up studies, the LMT/HMT value varied in parallel with tumor growth. The unimodal patterns of macrophages changed to multiple peaks with tumor burden, in experimental animals and in clinical study. At terminal stage, it showed unimodal peak of lower mobility and phagocytic activity decreased. The result indicates that LMT associated with depressed immune state of cancer patients, and electrophoresis of immuno-competent cells may be usefull for cancer diagnosis and early detection of recurrence, because this method is very easy to assay and highly reproducible.

[Diagnosis of cancer using lymphocyte electrophoresis (3): Electrophoretic mobility, interleukin 1 and prostaglandin E2 productions of monocytes in peripheral blood of cancer patients].

The S/F ratio of slow electrophoretic mobility cells (less than 0.95 micron/sec/V/cm) to fast mobility cells (greater than or equal to 0.95) increased significantly in the peripheral blood mononuclear cells of cancer patients. Since one of slow mobility cells was mainly composed of monocytes, the relationship between the mobility and the functions of monocytes in the peripheral blood of patients with cancer was examined. The cancer patients (total 27) consisted of 12 patients with rectum, 7 with colon, 3 with stomach, one with ovary and 4 with recurrent cancers and of 3 with stage 1, 11 with stage 2, 3 with stage 3 and 6 with stage 4 + 5. We determined the releases of interleukin 1 (IL-1) and prostaglandin E2 (PGE2) from monocytes, which were stimulated with lipopolysaccharide, as an index for the function of monocytes. The mobility of monocytes and productions of IL-1 and PGE2 from monocytes decreased with the stage of cancer patients, indicating a decreased function of monocytes in the patients. These findings suggest that S/F ratio of the peripheral blood mononuclear cells and mobility, IL-1 and PGE2 productions of monocytes are a good index for the judgement of treatment efficacy of cancer.

Effects of CD4+ and CD8+ T cells in tumor-bearing mice on antibody production.

The function of T cell subsets in tumor-bearing mice was examined using an in vitro culture system of anti-(sheep red blood cell) antibody production, which is known to be dependent on T cells. The helper function of T cells of fibrosarcoma-MethA-bearing mice in antibody production decreased with the tumor stage of the mice. T cells were separated into CD4+ and CD8+ cells for further analysis of T cell subsets by the panning method using monoclonal antibodies. The helper function of CD4+ T cells in antibody production began to decrease significantly in tumor-bearing mice 1 week after the tumor transplantation. On the other hand, the suppressive function of CD8+ T cells was retained and had not decreased in the mice even 3 weeks after the transplantation. The same changes in function of CD4+ and CD8+ T cells were also observed in Meth1-bearing mice. These results suggested that this tumor-associated immunosuppression in antibody production is attributable to the decrease in helper activity of CD4+ T cells and the maintenance of the suppressive activity of CD8+ T cells.