Murine Mammary Adenocarcinoma Research Articles

Inhibition of GSH-dependent PGH 2 isomerase in mammary adenocarcinoma cells by allicin.

We have reported tha allicin, a constituent of garlic oil, has no effect on the activities of platelet cyclooxygenase or thromboxane synthase, or vascular PGI 2 synthase. The effect of allicin on glutathione (GSH) dependent PGH 2 to PGE 2 isomerase is unknown. We therefore studied the effect of allicin on PGE 2 biosynthesis in a murine mammary adenocarcinoma cell line (No 4526). Intact or sonicated cells were incubated with either 14C-arachidonic acid (AA) or 14C-PHG 2, respectively. Following metabolism, products were extracted, separated by TLC and analyzed by radiochromatographic scan. PGE 2 was predominantly formed with minimal amounts of PGF 2α and PGD 2. Formation of 6-keto-PGF 1α or TXB 2 was not detected indicating the absence of TXA 2 and PGI 2 synthase activity. Indomethacin and ibuprofen inhibited the PGE 2 formation (p < 0.05). The enzymatic PGE 2 formation in sonicates was blocked by depletion of the cellular non-protein thiols by buthionine sulfoximine and was shown to be dependent on GSH. Allicin, over the range of 10–1000 μM, inhibited the formation of PGE 2 in cells exposed to 2.0 μM 14C-AA for 20 min. and in sonicated cells incubated with 20.0 μM 14C-PGH 2 for 2 min (p < 0.05). Allicin did not alter cyclooexygenase-mediated oxygen utilization in ram seminal vessicle microsomes, suggesting that allicin selectively inhibits the GSH-dependent PGH 2 to PGE 2 isomerase in this adenocarcinoma cell line.

Radiosensitizing effect of camphor on transplantable mammary adenocarcinoma in mice

The comparative radiosensitizing effects of camphor and metronidazole on murine transplantable mammary adenocarcinoma are reported. Male C 3 H Jax mice bearing transplanted mammary tumours were treated with camphor (0.5 μM/body wt) or metronidazole (0.5 μM/g body wt) 45 min before subjecting to local X-irradiation at the dose levels of 30, 80, 100 or 120 Gy. Sequential in situ measurement of the tumour volumes during the follow-up period of 45 days revealed that the maximum enhancement ratios of tumour growth delay for camphor and metronidazole were 4.8 and 2.5, respectively. This suggests that camphor can be a potential radiosensitizing agent in cancer radiotherapy.

Antitumor and toxicologic properties of the organometallic anticancer agent vanadocene dichloride

We report here on the antineoplastic, toxicologic, and transmembrane transfer properties of vanadocene dichloride (VDC), a representative metallocene dihalide. VDC is cytotoxic to HEp-2 human epidermoid carcinoma cells, in vitro, in a dose dependent manner, with a D o value (dose increment reducing the survival fraction by 1/e) of 0.530 ± 0.005 μg/ml. Under similar experimental conditions, the D o for cisplatin (CDDP) against these cells is 0.46 ± 0.08 μg/ml. In a murine mammary adenocarcinoma (TA3Ha) system, 125 μg/ml VDC inhibits the tumor-forming ability of 10 5 cells upon i.p. inoculation into syngeneic Strain A mice. The transmembrane transfer rate constants for the metal uptake of VDC and CDDP by TA3Ha cells in vitro were found to be 3.3 ± 0.8 × 10 −4 min −1 and 12 ± 2.0 × 10 t-4 min −1, respectively. In vivo studies with TA3Ha cells show that two i.p. treatments of 20, 40, and 60 mg/kg VDC increase the host survival by 30, 50, and 90%, respectively. Under similar conditions, 2, 4, and 6 mg/kg CDDP (equitoxic dose levels) prolong the host survival 50, 75, and 83%, respectively. Morphological, blood urea nitrogen level, and serum creatinine level data for Strain A mice treated with 60 mg/kg VDC give no evidence of renal or small intestinal damage. However, changes in the liver consistent with fatty cell degeneration are observed in these mice.

Tumor necrosis can facilitate the appearance of metastases.

The non-metastatic murine mammary adenocarcinoma M3 and its metastatic variant MM3 were used to evaluate the role of intratumoral necrosis in cell detachment and metastasis. Accelular extracts from necrotic areas of both tumors increased in vitro cellular detachment from M3 but not from MM3 fragments. Furthermore, the in vivo inoculation of the necrotic extracts within non-metastatic M3 tumors gave rise to pulmonary metastases. Histological studies revealed in M3 a central necrosis limited by an uninterrupted peripheral ring of well preserved cells, while in MM3 necrotic and non-necrotic areas alternated. It is concluded that the distribution of necrosis within the primary tumor by facilitating cell detachment is, at least in part, responsible for the development of metastases.

Heterogeneity and clonal interactions in the TS/A murine mammary adenocarcinoma.

Heterogeneity and clonal interactions in the TS/A murine mammary adenocarcinoma.

Establishment of murine macrophage hybridoma clones capable of acquiring tumoricidal activity upon activation with recombinant interferon-gamma and lipopolysaccharide.

Murine macrophage hybridoma clones were established by fusing glycogen-elicited peritoneal exudate cells (glycogen-PEC) derived from C3H/HeN mice and the hypoxanthine-aminopterin-thymidine-sensitive murine macrophage cell line, J774.3-2. The macrophage hybridomas were further screened for the capacity to acquire tumoricidal activity upon stimulation with lipopolysaccharide (LPS) and recombinant interferon-gamma (IFN-gamma) using murine mammary adenocarcinoma MM48 cells as targets, and three macrophage hybridoma clones, KM-1, KM-2, and KM-3, were established. With concomitant stimulation with LPS, IFN-gamma activated these hybridomas dose dependently to exhibit high tumoricidal activity, whereas single stimulation with either INF-gamma or LPS, even with higher concentrations, did not activate the macrophage hybridomas. This contrasted with the activation of glycogen-PEC for eliciting tumoricidal activity with a single stimulation with LPS (greater than 1 ng/ml) or IFN-gamma (greater than 10 IU/ml). Thus, the macrophage hybridoma clones established here represent inflammatory macrophages which require both IFN-gamma and LPS for their activation.

Rapid, quantitative microassay for the monokine respiration inhibitory factor

The murine monokine respiration inhibitory factor (RIF) induces lesions at Complex I and Complex II of the mitochondrial electron transport chain (ETC) of tumor cells; these lesions in the ETC appear closely linked with cytostasis of the targets. In this report we describe the use of the sensitive murine mammary adenocarcinoma line EMT-6 in a colorimetric microassay for the effects of RIF on the ETC and target replication. The participation of cytolytic molecules in this assay system was excluded because of the resistance of the target to their effects. The endpoint for the assay was the ETC-mediated reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its colored formazan. The two major coupling sites of MTT in the ETC of EMT-6 cells are shown to be proximal to Coenzyme Q, detected either by malate oxidation through Complex I or succinate oxidation through Complex II. The assay was sensitive to both RIF-induced lesions at these dehydrogenases and to the cytostasis-linked reduction in target cell number. The assessment of ETC lesions by this microassay correlated directly with that determined by the less sensitive polarimetric assay based on oxygen consumption. We demonstrate the application of this microassay to parameters for the production of RIF by activated murine macrophages, and to initial molecular characterization of this mediator.

Interactions of Natural Effector Cells and Prostaglandins in the Control of Metastasis&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

The roles of prostaglandins and nonspecific immune effector cells were examined in the growth and metastasis of a series of murine mammary adenocarcinomas growing in BALB/c mice. The spontaneous metastasis of the highly metastatic tumor designated 410.4 was inhibited by oral administration of the prostaglandin synthesis inhibitor indomethacin (INDO), whereas treatment of mice with the interferon inducer polyinosinic-polycytidylic acid (poly I:C) delayed tumor growth but did not affect the rate of spontaneous metastasis. Lung colony formation was dramatically reduced by poly I:C treatment following iv tumor cell inoculation (experimental metastasis), whereas oral INDO had no significant effect. Suppression of natural killer cell activity with anti-asialo GM1 (asGM1) antibody increased lung colony-forming ability of mammary tumor lines with either high, moderate, or low metastatic capacity. Although pretreatment of the recipient with INDO did not alter the incidence of experimental metastasis, culture of the tumor cells with INDO (1 micron) prior to iv inoculation inhibited the appearance of lung colonies by greater than 90%. This beneficial effect was dependent on an asGM1-positive population. These results suggest that in this murine mammary tumor system, prostaglandin synthesis contributes to metastatic capacity and that the beneficial effects of INDO may be attributable, in part, to the enhancement of tumor cell sensitivity to natural cytotoxic effector mechanisms.

Extraction of immunogenic murine mammary tumor antigens with 1-butanol.

Solubilized tumor associated antigens (TAA) were prepared by a single phase 1-butanol extraction of a transplantable murine mammary adenocarcinoma (H2712), other syngeneic non-mammary tumors, normal mammary tissue, and embryonic tissues. The crude butanol extract (CBE) of H2712 was found to have tumor-specific activity both in vitro in the leukocyte adherence inhibition (LAI) assay and in vivo in an immunoprophylaxis assay. The failure to detect any of the major mammary tumor virus (MTV) envelope glycoproteins or internal core protein in CBE-H2712 and the failure of tumor-bearing mice to give a LAI response to CBE prepared from embryonic tissues suggests that the CBE antigens which are responsible for LAI activity are not viral or oncofetal antigens. Enzymatic treatment of the CBE-H2712 indicated that a sialated glycoprotein formed the antigenic determinants recognized in CBE by the LAI assay. Gel filtration of LAI-reactive CBE-H2712 on a Sephadex G-75 column isolated both the in vitro and in vivo tumor-specific activity in a fraction pool. Polyacrylamide gel electrophoresis revealed the presence of a 67 kD subunit as the major species in the active fraction.

Sensitization of tumor target cells to cytolysis by murine macrophage cytolytic factor by drugs inhibiting DNA, RNA, and protein synthesis.

The mechanism(s) with which tumor target cells actively resist the lethal lesion induced by murine macrophage cytolytic factory (CF) has been probed by drug-induced sensitization of these cells. We have examined whether drug-induced sensitization is attributable to the action of these drugs on cellular DNA, RNA, or protein synthetic rates. The murine mammary adenocarcinoma cell line EMT-6 pretreated with a dose range of actinomycin-D (.03-3.0 micrograms/ml) for 4 hr was inhibited from 66 to 98% in DNA synthesis rate, from 81 to 93% in RNA synthesis rate, and from 38 to 52% in protein synthesis rate. The maximum sensitization toward lysis by CF was achieved with a drug dose of 1.0 micrograms/ml. The lack of correlation between the drug-induced effects on sensitization and effects on these metabolic rates was evident from the correlation coefficients for the percentage of maximum sensitization of the target toward CF-induced lysis, and the percent of inhibition of DNA, RNA, and protein synthesis. These were 0.11, -0.11, -0.44, respectively. Similarly, target cells treated with a dose range of cycloheximide (0.3-30 micrograms/ml) were inhibited from 81 to 95% in DNA synthesis rate, 68 to 81% in RNA synthesis rate, and 82 to 93% in protein synthesis rate. However, at none of the drug levels employed was significant sensitization of the target cell to CF-induced lysis observed. This was reflected in the correlation coefficients of -0.55, -0.28, and -0.34 for DNA, RNA, and protein synthetic rates, respectively. The essential role of cellular microtubule-dependent events early in the lytic mechanism was demonstrated by exposing colchicone-treated targets to CF. While colchicone could markedly inhibit lysis if introduced before CF, this inhibitory effect was not detected if the drug were withheld for 4 hr after CF exposure. The importance of the repair mechanism(s) early in the cellular response to lesion formation was demonstrated by altering the schedule for CF and actinomycin D administration to the target. If actinomycin D treatment was withheld from the targets for as little as 3 hr following introduction of CF, the lytic mechanism had already bypassed the drug-sensitive steps, manifest in markedly decreased susceptibility to lysis. Collectively, the data show that the ability of the EMT-6 target cells to resist CF-induced lysis does not depend solely on the ability of the cells to synthesize DNA, RNA, or protein.

Biochemical effects of dithiolthiones

Administration of dithiolthiones to mice in single intragastric doses (2–4 mmol/kg body weight) or in the diet (0.5% for 14 days), and to rats in the diet (0.1% for 14 days) was found to increase glutathione levels and the activities of a number of enzymes in various tissues including the liver and lung. The enzymes affected were glutathione transferases (with chlorodinitrobenzene or dichloronitrobenzene), quinone and glutathione reductase, and glucose-6-phosphate and 6-phosphogluconate dehydrogenase, all of which are involved directly or indirectly in the detoxication of xenobiotics, including carcinogens. The dithiolthiones tested in mice were oltipraz, ADT, 116L and 129L, and in rats, oltipraz. Intragastric administration of dithiolthiones (oltipraz, ADT or 116L; two doses each of 1 g/kg body weight) did not increase glutathione levels or enzyme activities in murine mammary adenocarcinoma transplants. Increases in glutathione levels and enzyme activities similar to those found with dithiolthiones were observed when a semi-synthetic diet containing 10–40% lyophilized cabbage was fed to mice for 30 days. Dithiolthiones that are present in cabbage may play a role in the protective actions of diets high in vegetables against the toxic actions of xenobiotics. The biochemical effects of dithiolthiones reported here may account for the protective actions of these compounds.

Redifferentiation of cancer cells: bestatin, estradiol, and prostaglandin D2.

The induction of redifferentiation of cancer cells was studied. This was achievable with bestatin, especially in combination with sex hormones or prostaglandin D2, in murine undifferentiated mammary adenocarcinoma cells in vitro. Prostaglandin D2 also exerted a similar redifferentiating effect on M1 murine leukemia cells and a consecutive bestatin-hormone regimen was effective in endocrine tumors in man. Cancer redifferentiation may one day be developed into a clinical technique.

Radioimmunodetection of murine mammary adenocarcinoma (TA3/Ha) lung and liver metastases with radioiodinated PNA

Organ-specific lung and liver metastatic variants of the murine TA3/Ha mammary adenocarcinoma cell line were selected by sequential in vivo growth with intervening in vitro cell culture. These variants readily formed specific lung or liver metastatic lesions upon i.v. injection into A/J mice. TA3/Ha cells produce a large cell surface glycoprotein called epiglycanin, which contains a high proportion of Thomsen-Friedenreich (TF) antigenic structures. The presence of non-cryptic TF has been associated with malignancy in humans and animals. We used peanut lectin agglutinin (PNA), which has a preferential affinity for TF antigenic structures, to determine whether these selected metastatic variants retained the TF antigen expression. In vitro, the TA3/Ha metastatic variant lines exhibited strong PNA binding similar to that seen with human RBC after neuraminidase treatment to expose the cryptic TF antigen. In contrast, the non-epiglycanin-producing TA3/St subline did not bind PNA appreciably. Autoradiography of liver sections with TA3/Ha metastatic lesions after 125I-PNA i.v. indicated an avid uptake throughout the viable tumor mass and FITC-PNA staining of these tissue sections readily identified the metastatic tumors under fluorescence microscopy. Tissue biodistribution studies revealed that lung or liver containing the TA3/Ha metastatic variant nodules retained about 7 to 8 times as much of an i.v. dose of radioiodinated PNA as did controls, allowing for clear delineation of tumor-infiltrated lung or liver by gamma scintigraphy. These in vitro and in vivo tests confirm that the selected organ-specific TA3/Ha variants retained the binding characteristics of the parent TA3/Ha line. These observations illustrate the potential utility of radiolabelled PNA for the detection of TF-antigen-expressing tumors and metastases. This murine system with organ-specific TA3/Ha metastatic variants also provides a model for evaluation of various other macromolecular probes for tumor radioimmunodetection of metastatic lesions.

Purification and characterization of platelet aggregating activity from tumor cells: Copurification with procoagulant activity

The platelet aggregating component from murine 15091A mammary adenocarcinoma cells was purified by solubilization of activity with CHAPS (3-[(3-cholidamidopropyl)-dimethylammonio]-1-propane sulfonate), fractionation with ammonium sulfate, ion exchange chromatography on DEAE cellulose, and hydrophobic interaction chromatography on dodecyl agarose. A purification of 90–100 fold over the initial cell homogenate was achieved. SDS-PAGE of the purified material resulted in a single major band with a molecular weight of 51,000 = 2,000. Procoagulant activity was found to copurify with platelet aggregating activity. Reconstitution with phospholipids was necessary to obtain platelet aggregating activity and procoagulant activity. Trypsin abolished both platelet aggregating and procoagulant activities. The irreversible proteinase inhibitors phenylmethylsulfonyl fluoride, Nα-p-tosyl-L-lysine chloromethyl ketone, iodoacetamide or phenanthroline had no effect on platelet aggregating or procoagulant activities. Platelet aggregation induced by this material was inhibited by low concentrations of the specific irreversible thrombin inhibitors, dansylarginine N-(3-ethyl-1,5-pentanediyl) amide and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone. This is the first report of copurification of tumor cell platelet aggregating and coagulating activities.

Hyperbaric Pressure Effects Measured by Growth of a Transplantable Tumor in the C3H/HeN Mouse&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

Both hypobaric exposure at 0.5 atmospheres absolute (ATA) and hyperbaric pressure exposure at 3.5-8 ATA slowed transplantable tumor growth. These experiments detailed the hyperbaric pressure exposure. C3H/HeN-MTV+ mice, bearing the 16/C transplantable murine mammary adenocarcinoma and exposed to 18 days' treatment by a hyperbaric chamber at 3.5-8 ATA, had tumor weights that averaged 50-75% less than the tumor weights in mice caged at ambient ("sea level") pressure. A series of experiments was run to investigate this response to hyperbaric pressure exposure. After mice underwent continuous exposure to 3.5-8 ATA normoxic (normal oxygen) hyperbaric pressure with use of either argon or nitrogen inert gas, which began 3 days after tumor inoculation, tumors were removed at about 3 weeks' growth from these pressure-exposed mice and measured for growth by weighing. Final tumor weight in pressure-exposed experimental mice was significantly less than tumor weight in paired groups of tumor-bearing controls that received no hyperbaric pressure. Tumor weight was inversely related to pressure "dose," although the small pressure range produced an effect at all pressures used. The number of compression-decompression cycles to which the animals were subjected, however, was related positively to tumor weight at necropsy. Continued tumor growth in mice subjected to frequent pressure change (in conjunction with pressure exposure that otherwise limited tumor size) was unexplained by these experiments. The greatest difference between tumor weights in controls and pressure-exposed animals was seen with 2 weeks' continuous pressure exposure. A limited profile of blood tests was performed, and these reflected only minor, expected change in the pressure-exposed experimental animals. The data at hand did not suggest a mechanism by which chronic normoxic hyperbaric pressure limited tumor size.

Immunological responses to a murine mammary adenocarcinoma. Outgrowth of pulmonary occult metastases induced by immunosuppressive perturbations.

Intravenously (i.v.) or subcutaneously (s.c.) injected T1699 tumor cell subpopulations (Ts, and TLI-1) were destroyed more rapidly in BALB/c nu/nu athymic mice than in syngeneic DBA/2J mice. The number of artificial pulmonary metastases of TLI-1 tumors was significantly lower, and the growth rate of s.c. Ts, and TLI-1 tumors was correspondingly slower in the nude mice. No macroscopic outgrowth of pulmonary metastases of Ts tumors was detected in either group of s.c. tumor-bearers, even though the s.c. tumors progressed and killed the hosts. Unexpectedly, however, s.c. implantation in normal DBA/2J mice of lung homogenates not only from DBA/2J but also from s.c. Ts tumor-bearing BALB/c nu/nu mice produced tumors, suggesting that significant numbers of clonogenic Ts cells were present in the lungs of animals without spontaneous outgrowth of pulmonary metastases. Perturbations of s.c. Ts tumor-bearing DBA/2J or BALB/c nu/nu mice with immunosuppressive drugs or with anti-mouse thymocyte serum, respectively, induced rapid outgrowth of pulmonary metastases.

Heterogeneity of murine mammary adenocarcinoma cell subpopulations. In vitro and in vivo resistance to macrophage cytotoxicity and its association with metastatic capacity.

Properties of Ts, a long-term tissue culture line of T1699 mammary adenocarcinoma of DBA/2 mice, and two of its sublines - TR2 and TLI-1-were comparatively studied. Ts tumors produced no spontaneous metastases, nor did i.v. injection of up to 1 X 10(6) Ts cells produce a lung tumor nodule. Ts cells were susceptible to macrophage cytotoxicity in vitro and i.v. injected cells were rapidly destroyed in the lungs. TLI-1 tumors spontaneously metastasized to the lungs, and i.v. injection of 1 X 10(3) TLI-1 cells produced approximately 15 lung nodules per animal. TLI-1 cells were least susceptible to both macrophage-mediated cytolysis in vitro and in vivo host antitumor mechanisms. TR2 cells were intermediate with respect to all these properties. Differences in their susceptibility to macrophage cytotoxicity were recognized not only by normal peritoneal macrophages but also by murine macrophage lines. On the other hand, all the subpopulations were uniformly resistant to NK activity in vitro. These findings suggest that resistance of tumor cells in vitro to macrophage cytotoxicity corresponds with their in vivo metastatic potential.

Studies of mammary carcinoma metastasis in a mouse model system. I: Derivation and characterization of cells with different metastatic properties during tumour progression in vivo.

The biological and metastatic properties of cells from a murine mammary adenocarcinoma, MT1, were studied during serial transplantation in syngeneic hosts. Over 35 generations the tumour progressed from a well-differentiated, poorly metastatic neoplasm to an anaplastic highly metastatic state. At early passages the tumour yielded uniform cultures of cuboidal epithelial cells, at passage 17 both epitheloid and spindle type cells were present, and by passage 30 only spindle type cells were obtained. Epithelioid cell lines and clones when injected intravenously into syngeneic hosts produced lung colonies only, whereas spindle cell lines were capable of extensive extrapulmonary colonisation. Similar patterns of dissemination and growth were seen in spontaneous metastasis assays. In spite of the marked phenotypic differences in these 'subpopulations', their comparable ultrastructural features, oestrogen receptor levels, expression of MMTV antigens, DNA content and lectin binding profiles suggested a common cell lineage. It is proposed that these cell lines will be of use in the determination of tumour and host factors influencing tumour progression and the evolution of metastatic potential.

Consecutive bestatin-hormone regimen induces redifferentiation of cancer cells in vitro.

When undifferentiated murine mammary adenocarcinoma cells were consecutively exposed to bestatin and estradiol or testosterone in vitro, morphological signs of redifferentiation of the cells were observed along with cellular enlargement and subsequent cell loss. These observations suggest that similar treatments can be applied clinically to cancers of the breast and prostate.

Atmospheric Pressure Effects on Tumor Growth: Hypobaric Anoxia and Growth of a Murine Transplantable Tumor23

Inasmuch as solid tumor growth and some intervention methods for tumor control have often been related to the low oxygen levels in tumor tissue, and a special role for hypoxia, perhaps even in oncogenesis, has been suggested by observations of unexpectedly low tumor incidence in mice caged a lifetime in the environment of a simulated altitude, inbred C3H/HeN mammary tumor virus-positive mice bearing transplanted tumors (16/C murine mammary adenocarcinoma) were exposed to atmospheric pressure variants ranging from 0.33 to 2.0 in different sequences 24 hours/day. Breathing gases included air, 100% oxygen, and other nitrogen--oxygen combinations. Exposure to the pressure sequences was continuous, beginning on the third day after tumor inoculation and continuing until planned necropsy at 1, 2, or 3 weeks. Actual tumor weight was used as a measure of effect. Mice caged at simulated altitude had tumors that averaged 45% of the weight of control tumors. The maximum effect occurred with continuous 2-week exposure to 0.43 atm. beginning on day 3 of tumor growth. At necropsy, these experimental tumors weighed an average of 15% of the control tumor weight. Life-span studies showed a maximum of 36% increase in longevity in the hypobaric pressure-exposed mice when compared to that of unexposed controls.