Rat Fibrosarcoma Research Articles

抗癌剤による腫よう関連抗原のｓｈｅｄｄｉｎｇ抑制と機序解析

We have previously reported that the 3-methylcolanthrene-induced rat fibrosarcoma KMT-17 cell line and its subclone A3 shed a tumor-associated antigen (TAA), termed CE7, from the cell surface and that this phenomenon might play a role in an immunological escape mechanism. We also reported that CE7-antigen shedding was inhibited by low dose (30 Gy) X-irradiation in vivo or in vitro, the antigen was expressed on the cell surface and the immunogenicity of CE7-antigen expressing cells was enhanced. The purpose of this study was to examine whether this CE7-antigen shedding is inhibited by the antitumor d rugs bleomycin (BLM), mitomycin C (MMC) and cyclophosphamide (4H-CY) and to clarify the mechanism of antigen shedding. We found that CE7-antigen shedding was inhibited following treatment with these antitumor drugs in vitro. When FCS concentration in media was changed, CE7-antigen expression was enhanced parallel to the extent of inhibiti on of cell growth (prolonged doubling time). We demonstrated CE7-antigen in A3 cells grown in 10% FCS medium by Western blotting. A large amount of CE7-antigen was localized in the cytoplasm of the cells. These results suggest that A3 cells in 10% FCS medium produce CE7-antigen, store it in the cytoplasm and shed it from the cell surface in vesicular form and that antitumor drug treatment inhibits vesicle production, enhancing CE7-antigen expression on the cell surface.

Deuterium nuclear magnetic resonance imaging of the developmental pattern of tumour blood flow.

31P Magnetic Resonance Spectroscopy has been used by various workers to study tumour bioenergetic status and also to monitor tumour response to various therapeutic modalities such as chemotherapy, radiotherapy and hyperthermia. Many of the observations reported may be explained by changes in tumour perfusion and oxygenation [1–3]. Recently non-invasive 2H NMR spectroscopy techniques, measuring uptake or clearance of D20, have been introduced, thus enabling repetitive estimation of tumour blood flow [4, 5]. Also, imaging the distribution of uptake of this stable, flow-limited tracer provides an indication of vascular competency across a tumour [6]. In the present study we have combined 2H imaging of D20 uptake with localized 31P spectroscopy to make serial measurements of tumour blood flow and bioenergetic status during the growth of a transplantable rat fibrosarcoma in order to examine whether there are significant changes in metabolism and perfusion as a tumour enlarges.KeywordsTumour Blood FlowHigh Energy PhosphateSolenoid CoilIncrease Tumour VolumeBioenergetic StatusThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The potential for prazosin and calcitonin gene-related peptide (CGRP) in causing hypoxia in tumours

Using 31P NMR spectroscopy, changes in tumour metabolic status were studied in a transplanted rat fibrosarcoma following the administration of vasodilators. Mean Arterial Blood Pressure (MABP) was monitored simultaneously. Two vasodilators were studied, prazosin and CGRP, which altered the NMR parameters Pi/sigma P, beta NTP,Pi, PCr/Pi and PME/Pi in a dose dependent manner. There was a good correlation between the various NMR parameters; for analysis, Pi/sigma P was used for convenience. With increasing doses of vasodilator, Pi/sigma P increased and the MABP decreased. Reduction in pHNMR showed a correlation with decreasing MABP following the administration of prazosin but not after CGRP. Both prazosin and CGRP produced changes in 31P NMR spectra consistent with a reduction in tumour blood flow. The results for prazosin and CGRP were comparable and showed a 15-20% increase in Pi/sigma P for a 20% reduction in MABP. These results were compared with those from hydralazine. With hydralazine an acceptable reduction in blood pressure (up to approximately 25%) has little effect and may even alter NMR parameters consistent with an increase in blood flow, a reduction of approximately 40% is required for a significant decrease in flow. Both prazosin and CGRP are shown to be far more effective than hydralazine in causing tumour hypoxia at a clinically acceptable reduction in blood pressure. CGRP may be the more suitable for clinical use because of its short half life, its capability to achieve controlled hypotension and the relatively few side effects associated with its use.

The improved effects of specific active immunotherapy on a rat fibrosarcoma by antitumor drugs.

We have tried to find out if the combination of a xenogenized tumor cell vaccine and antitumor drugs is able to induce a synergistic increase in the antitumor therapeutic effect. The degree of increase in the LTD50 (50% lethal tumor dose) is expressed numerically, as a quantitative index designed to compare degrees of transplantation resistance to tumor cell challenge. A LTD50 was achieved by an intradermal (i.d.) immunization with xenogenized tumor cells when challenged with tumor cells implanted intraperitoneally 2 weeks after the immunization: this LTD50 value was 527,000 times higher than that of the non-immunized group. When we combined this type of immunization with appropriate doses of bleomycin (BLM) or cyclophosphamide (CY), which are able to augment antitumor immunity, the LTD50 was 723,000-1,190,000 times higher than that of the non-immunized group. This increase in the LTD50 is definitely higher than that achieved by a single immunization with irradiated tumor cells (x 33,000) and combined with either BLM (x 93,000) or CY (x 140,000). We also studied the therapeutic effect of a tumor cell vaccine combined with antitumor drugs BLM or CY in tumor-bearing rats. We observed a synergistic effect caused by BLM or CY after i.d. immunization with xenogenized tumor cells: this showed a significant increase when compared with the therapeutic effects obtained by chemotherapy alone (P less than 0.01). Nevertheless, there was no evidence that the above antitumor effects is superior to the effect achieved by irradiated tumor cells.

The morphology, deformability and microvascular arrest of rat fibrosarcoma and adenocarcinoma cells

The deformation and flow properties of tumour cells may play a role in their arrest in the microvasculature of different organs. In the present investigation the morphology, deformability and microvascular arrest in the liver of rat fibrosarcoma cells (FSCs) and adenocarcinoma cells (ACCs) were compared using electron microscopy, deformability measurements in narrow glass pipettes and isotope-labelling techniques. The ACCs had a larger mean diameter (13.9 microns) than the FSCs (10.9 microns) and showed a slower rate of deformation into 6.5 microns glass pipettes. A significantly larger percentage of ACCSs (52.4%) than of FSCs (19.9%) remained in the livers 5 min after intraportal injection. The results indicate that for the particular tumour cells studied here, there exists a relationship between cell deformability and the tendency for microvascular trapping in the liver, i.e. less deformable cells have a greater tendency for retention in the liver.

Follow-up by 31P NMR spectroscopy of the energy metabolism of malignant tumor in rats during treatment

The energy metabolism of tumors in rats was investigated by in vivo 31P- NMR spectroscopy. The effects of radiotherapy, chemotherapy or radiotherapy combined with 5-fluorouracil (5-FU) chemotherapy were evaluated by observing the changes of these spectra in chemically induced subcutaneous fibrosarcoma in rats. Two milligrams of DMBA in solution in olive oil were administered subcutaneously in the flank of 20 Wistar rats and 17 fibrosarcoma occurred. 13P NMR spectra were recorded with a Broker Medspec 30 47 spectrometer using a surface coil positioned over the tumor. We did not observe significant changes in the spectra during tumor growth. Radiotherapy and 5-FU chemotherapy alone did not induce major changes in the 31P spectra. But the situation was completely different for animals receiving the therapeutic combination. A clear increase in the ratio of inorganic phosphate to total phosphorus signal was observed 48 h after the first irradiation session. The pH shifted concurrently to the acidic range. No effect on tumor regression was observed in the rats from the chemotherapy group, while regression was less than 50% in rats treated by irradiation only, and at least 80% in the combined group.

A comparison between morphological, rheological and lodgement properties of rat fibrosarcoma cells harvested from solid tumours and cultures

Vital microscopic studies on tumour cell lodgement in the rat liver have suggested that tumour cells from culture (CTCs) and from solid tumour (STCs) have different rheological properties. In the present study CTCs and STCs from a rat fibrosarcoma were compared in respect to their morphology, rheology and lodgement properties. The ultrastructural examination showed that the CTCs had a larger mean diameter (14.9 microns) than the STCs (11.2 microns). Both cell types had a very irregular nucleus. Surface irregularities provide the CTCs and STCs with a 'membrane excess', i.e. a larger plasmalemma than required to enclose the cells as smooth spheres, amounting to 46.8% and 60.9%, respectively. These values indicate that both CTCs and STCs can be substantially deformed with preservation of the volume and area. The CTCs were stiffer than the STCs when deformed at constant pressure in 6.5 microns glass pipettes, the nucleus appearing to be a significant hindrance to CTC deformation. Five minutes after intraportal injection of radiolabelled CTCs and STCs a much higher percentage of CTCs (82%) than of STCs (20%) remained lodged in the rat liver. The results indicate that the propagation in culture of fibrosarcoma cells alters the morphology and rheology of the cells such that CTCs are not suited for studies in vivo where the spontaneous intravascular dissemination and organ lodgement of tumour cells is simulated.

Isolation and characterization of a serologically defined tumour membrane antigen from a chemically-induced rat sarcoma, HSN

Polypeptides containing the tumour antigenic determinant present on the external domain of a membrane antigen of the 3-4 benzpyrene-induced rat fibrosarcoma HSN have been isolated and purified. Following cleavage from intact cells with trypsin, the peptides were purified by immunoaffinity chromatography and SDS-PAGE. Three polypeptides of molecular weight 120, 45 and 42 kDa were obtained that bound the specific rat monoclonal antibody (MAb) 11/160 in Western blots. Chemical analyses of the 45-kDa fragment yielded the following sequence: NH2-I V F P H G S L M V I L E H T Q K P All 14 of the syngeneic MAbs prepared from rats bearing the HSN tumour competed with each other to bind to the specific antigen. Western blots of the purified tryptic fragments probed with 125I-AbI revealed that, while some of the MAbs (11/160, ALN/12/17 and ALN/9/94) recognized a sequential determinant, others (ALN/11/53, ALN/16/53, and AL/3/12) bound to a conformational epitope. It is concluded that the AbI bind to distinct but overlapping epitopes.

Radiation-induced Modification of Blood Flow Distribution in a Rat Fibrosarcoma

Radiation-induced Modification of Blood Flow Distribution in a Rat Fibrosarcoma

Solid tumour chemotherapy using implantable collagen-poly (HEMA) hydrogel containing 5-fluorouracil

Implantable collagen-poly(HEMA) hydrogels containing the anticancer drug 5-fluorouracil (5-FU) have been prepared and evaluated for their efficacy towards a solid tumour fibrosarcoma in Wistar rats. The tumour was developed by inoculation of a 10% tumour-cell suspension in the anterior aspect of the hind limb. Four groups were studied--untreated control, intratumoural injection of free 5-FU, subcutaneous implantation of placebo and implantation of 5-FU-bearing hydrogel pellets (10 mm diameter x 1 mm thick) containing the drug in close proximity to the tumour. The hydrogel showed an improved antitumour activity over free 5-FU as evidenced by the gross tumour weight assessments and by [3H]thymidine incorporation in-vitro. This was attributed to the controlled and slow release of 5-FU compared with free 5-FU over the same period of treatment. The implantation of hydrogel could thus be a potential alternative to free 5-FU therapy in the treatment of solid tumours such as fibrosarcoma.

The influence of different flow velocities on tumor cell survival in micropore filters

Earlier studies have shown a lower degree of lodgement and early survival of tumor cells in muscle than in liver after infusion via the femoral artery and portal vein, respectively. A possible explanation to this difference might be that the tumor cells are mechanically destroyed, and thus die more rapidly in muscle because they enter this capillary network at a much higher flow velocity. In the present study, the effect on early tumor cell (rat fibrosarcoma) survival of a high and a low flow velocity/deformation rate was evaluated in micropore (5 microns) filters, using isotope (Cr51) technique. These experiments were combined with scanning electron microscopic (SEM) analyses. The filter experiments showed no significant differences in the rate of cell death in the filters between tumor cells subjected to high or low deformation rates, and there were no qualitative differences in tumor cell appearance in the SEM study. It is, therefore, concluded that the difference in tumor cell lodgement and survival between muscle and liver is not primarily caused by differences in the rate of cell deformation upon entry of the organ capillary network.

The existence of an inactive form of transglutaminase within metastasising tumours

Separation by anion exchange chromatography of detergent extracts from a poorly metastatic HSV-2-induced hamster fibrosarcoma, its highly metastatic variant and a highly metastatic rat fibrosarcoma indicated the presence of an inactive form of transglutaminase antigen, when eluent fractions were assayed for transglutaminase activity and antigen. This inactive antigenic transglutaminase was clearly separable from the particulate and cytosolic forms of the transglutaminase enzyme. Unlike tumours, its presence could not be demonstrated in extracts from normal rat liver. Measurement of activity levels during tumour growth indicated that the progression of the two highly metastatic tumours was accompanied by a decrease in cytosolic transglutaminase activity, whilst the activity of this enzyme form remained constant in the poorly metastatic tumour. Measurement of antigen levels indicated an inverse relationship between the level of inactive transglutaminase and the level of cytosolic transglutaminase activity, suggesting that the two forms are inter-related. Gel filtration indicated the molecular weight of the inactive form to be greater than both the particulate and cytosolic forms, and it was estimated to be 120 000. Partial proteolysis of the semi-purified inactive form, by either trypsin or thrombin, led to its activation and to the appearance of a transglutaminase similar in molecular weight and ionic mobility, both by anion-exchange chromatography and electrophoresis, to the cytosolic transglutaminase.

Blood flow and blood volume in a transplanted rat fibrosarcoma: comparison with various normal tissues

Blood flow measurements following i.v. infusion of iodo-antipyrine labelled with 14C( 14C-IAP) and blood volume measurements following i.v. injection of 125I human serum albumin and 51Cr-labelled red blood cells were made in a transplanted rat fibrosarcoma for comparison with various normal tissues. The tumour-blood partition co-efficient for 14C-IAP was found to be 0.79 ± 0.07 which is similar to most of the normal tissues studied. The solubility of 14C-IAP in plasma was found to be higher than that in whole blood. Blood flow to tumours < 1000 mm 3 was found to be 17.9 ± 4.0 ml blood 100 g tissue −1 · min −1. These values were considered to be primarily measurements of nutritive flow. Blood in the tumours was found to occupy around 1% of the tissue space which was similar to that found for normal muscle and skin. There was no direct correlation between % blood volume and blood flow for the different tissues studied. The haematocrit of blood contained in tumour tissue was calculated to be significantly lower than that of blood contained in the normal tissues. It was suspected that permeability of tumour blood vessel walls to 125I-HSA could have accounted for this difference.

The relationship between regional variations in blood flow and histology in a transplanted rat fibrosarcoma

The regional distribution of blood flow to the LBDS1 fibrosarcoma, transplanted into the subcutaneous site in rats, was investigated using the readily diffusible compound 14C-iodo-antipyrine (14C-IAP). Quantitative autoradiography was used to establish absolute values of specific blood flow F for 100 X 100 X 20 microns adjacent tissue volumes of the unperturbed tumour. Mean blood flow to whole tumours was found to decrease with increase in tumour size. This relationship was abolished if blood flow was only measured in sections cut from the periphery of the tumours. Detailed analysis of a sub-group of tumours showed that blood flow to individual tumours was heterogeneous. The range of blood flow was large, indicating that mean blood flow to a whole tumour is a poor reflection of the blood perfusion pattern of that tumour. Necrotic tumour regions were usually very poorly perfused. With the exception of the smallest tumours studied, blood flow was lower in the centre of tumours than in the periphery. Necrosis also tended to develop centrally. However, the peripheral to central gradient of blood flow was apparent even when densely cellular, viable tumour regions and necrotic regions were analysed separately. The decrease in blood flow with tumour size was also apparent in densely cellular, viable tumour regions when analysed separately. Qualitative comparison of tumour histology and regional blood flow showed that there were areas of very low blood flow associated with viable tumour regions. Less common were areas of rather high blood flow associated with necrotic tumour regions. A complicated relationship exists between tumour histology and blood flow. The quantitative autoradiography technique is suitable for investigating the most poorly perfused and the most well perfused viable fractions of animal tumours which may limit the efficacy of different types of therapy.

放射線照射による腫よう関連抗原のｓｈｅｄｄｉｎｇ抑制に関する研究

We have previously reported that the 3-methylcolanthrene induced rat fibrosarcoma KMT-17 cell line and its subclone A3 shed a TAA, termed CE 7, from the cell surface and that this phenomenon might play a role in the immunological escape mechanism.We have also reported that, when tumors in rats were treated by local 30 Gy irradiation, anti-tumor immune responses of spleen cells obtained from these treated rats were augumented. Thus, we examined whether this CE 7 antigen shedding is inhibited by x-irradiation and as a result tumor immunogenicity is enhanced. When A 3 cells growing in a medium containing 10% FCS were irradiated with 60Co-γ-rays on day 2, the expression of CE 7 antigen on A 3 cells was enhanced. These CE 7 expressing cells were fixed with 1 % paraformaldehyde and used for immunization by i. d. inoculation into syngenic WKA rats. The immunogenicity of CE 7 expressing cells was increased at least 100 fold (increase of LTD50) as compared to non-irradiated cells. KMT-17 cells were transplanted into the right hind limb of syngenic WKA rats and the local tumor area was treated by irradiation with 10, 30 or 60 Gy 5 days after transplantation. CE 7 antigen positive cells were found in greater proportion within 30 Gy-irradiated tumors than in nonirradiated, 10 Gy-irradiated or 60Gy-irradiated tumors. These results suggested that irradiation enhanced the CE 7 antigen expression on A 3 and KMT-17 cells and that these CE7 antigen expressing cells can stimulate the anti-tumor immunity.

Excision repair differences in rat fibroblasts

By using a cellular transformation system, which has been developed by Graffi et al., we analysed differences in the excision capacity of UV-induced dimers in normal cells, in vitro transformed cells and in malignant cells derived from a rat fibrosarcoma. The results suggest that both the transformed and the tumour-derived cells exhibit a kinetic pattern of excision repair different from that of the normal cells. Recognition of the dimers and the incision rate are more complete in the normal cell.

Phorbol ester-inducible T-cell-specific expression of variant mouse mammary tumor virus long terminal repeats

Acquired proviruses of mouse mammary tumor virus (MMTV) in T-cell leukemias of male GR mice have rearrangements in the U3 region of their long terminal repeats (LTR). In contrast to the endogenous nonrearranged MMTV proviruses, these mutated copies are highly expressed in leukemic T cells. To investigate whether the sequence alterations in the LTR are responsible for the high expression of rearranged MMTV proviruses, we made constructs in which normal and variant LTRs drive the bacterial reporter gene chloramphenicol acetyltransferase (CAT). Two different rearranged LTRs were used, one containing a 420-base-pair (bp) deletion (L13) and another carrying a 456-bp deletion plus an 82-bp insertion (L42). These constructs were transfected into murine (GRSL) and human (MOLT-4) T-cell lines that either had or had not been treated with phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]). In GRSL cells, the L13-LTR-CAT construct showed transcriptional activity that was further enhanced by TPA. In MOLT-4 cells, both variant LTRs were active, but only after stimulation with TPA. In contrast, normal(N)-LTR-CAT constructs were not expressed, irrespective of TPA addition. In XC rat fibrosarcoma cells, neither normal nor variant LTRs gave rise to detectable CAT activity, either in the presence or in the absence of TPA, but dexamethasone strongly stimulated CAT activity driven by N and L42 LTRs. The L13 LTR was considerably less active, probably caused by the deletion of the distal part of the glucocorticoid responsive element. We conclude that the LTR rearrangements generate TPA responsiveness and contribute to T-cell-specific expression of MMTV variants.

Studies of phospholipase and cyclooxygenase activities in herpes simplex virus type 2-transformed rat cells

In this report we describe results showing that both arachidonic acid (20:4) release and prostaglandin (PG) synthesis in herpes simplex virus type 2 (HSV-2)-transformed tumor-derived rat fibrosarcoma (RFS) cells are inhibited by indomethacin (cyclooxygenase inhibitor). These data suggest that cyclooxygenase (prostaglandin endoperoxide synthetase, PES) and phospholipase are coupled in their regulation in RFS cells. Data obtained using a cell-free assay to measure directly cyclooxygenase specific activity in the absence of phospholipase activity indicate that constitutive cyclooxygenase activity is very low in non-transformed REF and is not induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, after an initial degradation of constitutive enzyme, cyclooxygenase is synthesized de novo following TPA treatment of HSV-2-transformed RFS clonal cells. At least a 5-fold induction of cyclooxygenase occurs by 6 h post-treatment of HSV-2-transformed RFS clonal cells. This induction is inhibited by pretreatment of cells with cycloheximide (protein synthesis inhibitor) 1 h prior to TPA treatment. Results obtained following delayed addition of cycloheximide to cells after TPA treatment indicate that the newly synthesized cyclooxygenase has a half-life of approximately 5 min. In addition, HSV-2-transformed RFS clonal cells at low passage (less than 10) show over a 3-fold greater increase in cyclooxygenase specific activity after TPA treatment compared to that in TPA-treated high passage (greater than 20) cells, suggesting that induction of high levels of PG synthesis is transient in these cells. These data indicate that HSV-2 transformation of REF cells is associated with alteration of cyclooxygenase regulation.

Cellular Heterogeneity: Explanation for Changing of Tumor Phenotype and Biologic Behavior in Soft Tissue Sarcomas

Many human soft tissue sarcomas are known to show variable structures in a given tumor and to be able of changing the histological feature during the course of the disease. These facts prompted an experimental study in order to ascertain if by use of long-term cultures of an established rat fibrosarcoma cell line (RFS) in respective inoculation tumors a histological picture different from the original fibrosarcoma could be produced. It was found that inoculation tumors generated by later subcultures partly corresponded to tumors with features of malignant histiocytomas and malignant fibrous histiocytomas. In a further experiment we attempted to initiate metastatic dissemination of a primarily non-metastasizing xenotransplanted RFS sarcoma in nude mice. By repeated diminution operations comparable to inadequate tumor surgery in human metastasis formation in lung parenchyme was attained. Taking into account cellular heterogeneity as basically responsible for the phenomena observed, different causal aspects concerning the experimental results are discussed. We deduce from our findings that the concept of histogenetic classification of soft tissue sarcomas should be replaced by an interpretation according to the actual differentiation. This also applies to malignant fibrous histiocytomas which are at least in part mesenchymal tumors and not descendants of mononuclear phagocytic cells and may originate by a dedifferentiation process of otherwise defined sarcoma types.

Hyaluronate appears to be covalently linked to the cell surface.

The purpose of this study was to examine the nature of the linkage between cell-surface hyaluronate and the plasma membrane. To accomplish this, rat fibrosarcoma cells were cultured in the presence of [3H]-acetate to isotopically label the hyaluronate, and then fixed with glutaraldehyde, which cross-links proteins but does not react directly with hyaluronate. The glutaraldehyde fixation stabilized the cells so that they could be manipulated in ways which would otherwise destroy cells. The fixed cells were then subjected to various treatments, and the amount of hyaluronate remaining on the cell surface was assayed via exhaustive digestion with Streptomyces hyaluronidase. Using this technique, we found that 1) cell-surface hyaluronate was quite stable for extended periods of time even in the presence of a large excess of non-labeled hyaluronate; 2) 4 M guanidine HCl and detergents did not extract a significant portion of cell-surface hyaluronate; 3) solutions of varying ionic strength (0-1 M NaCl) had no effect on the retention of hyaluronate; 4) the cell coat was stable in the range of pH 4-11, but outside this range a significant amount of hyaluronate was released; and 5) treatment with proteases released cell-surface hyaluronate. These results are consistent with the possibility that hyaluronate is covalently linked to a protein associated with the plasma membrane. Further support for this model came from experiments with the detergent Triton X-114, which can be used to separate soluble proteins from hydrophobic proteins. When nonfixed rat fibrosarcoma cells were extracted with this detergent and then partitioned by centrifugation, approximately 30 times as much hyaluronate was present in the detergent fraction which contained the hydrophobic proteins, as compared to the extracts pretreated with trypsin prior to phase separation. Again, these results suggest that cell-surface hyaluronate is directly linked to a hydrophobic core protein intercalated in the plasma membrane.