Antiproliferative and cytotoxic effects of different type cytostatics on mouse pluripotent stem and teratocarcinoma cells

Pluripotent stem cells are able to proliferate indefinitely and differentiate in vitro into various cell types. However, in most cases in vitro differentiation of the pluripotent stem cells is asynchronous and incomplete, and the residual undifferentiated cells can initiate teratoma development after transplantation into recipients. These features of the pluripotent stem cells are the major issue for development of safe cell therapy technologies based on pluripotent stem cells. Considering significant resemblance of growth rates of pluripotent stem and cancer cells we investigated antiproliferative and cytotoxic effects of different type cytostatics (mitomycin C, etoposide, vinblastine and cycloheximide) on the undifferentiated and differentiating mouse embryonic stem cells, embryonic germ cells, blastocyst and on mouse embryonal teratocarcinoma cells and mouse embryonic fibroblasts. The findings showed that all cytostatics used induced both antiproliferative effects and acute toxic processes in undifferentiated pluripotent stem cells and embryonal teratocarcinoma cells whereas these effects were less in differentiating embryonic stem cells and embryonic fibroblast. Moreover, the trophoblast cells of mouse blastocysts were less sensitive to damaging effects of cytostatics than inner cell mass cells. The examination of deferred effects of cytostatics revealed that the effects of mitomycin C, etoposide and vinblastine, but not cycloheximide, were irreversible because survived cells were not able to proliferate. Nevertheless, the numbers of embryonic fibroblasts exposed to etoposide or vinblastine remained unchanged while vast majority of undifferentiated pluripotent cells treated underwent apoptosis. Thus, diverse effects of etoposide and vinblastine on the undifferentiated pluripotent stem cells and differentiated embryonic cells allow us to consider these cytostatics and their analogs as drug-candidates for selective elimination of the residual undifferentiated pluripotent stem cells from population of differentiating cells. These findings demonstrate for the first time the possibility of selective elimination of undifferentiated pluripotent stem cells using cytostatic drugs approved for clinic practice. However, to improve effectiveness and safety of this approach and to prevent mutagenic, carcinogenic and teratogenic effects on undifferentiated pluripotent stem cells and their differentiated cell derivatives large-scale studies of cytostatic effects using different experimental design and active doses must be performed.     

Sister chromatid exchange studies for monitoring DNA damage and repair capacity after cytostatics in vitro and in lymphocytes of leukaemic patients under cytostatic therapy.

The effect of various cytostatic drugs was studied on the frequency of sister chromatid exchanges (SCEs) in vitro and in PHA-stimulated lymphocytes of leukaemic patients under cytostatic therapy. The lymphocyte system is a sensitive one for the detection of DNA damage after administration of cytostatic drugs in vitro. Mitomycin C, busulphan, vincristine, chlorambucil, cytosine arabinoside, cyclophosphamide and lycurim were tested. All except cyclophosphamide induced high frequencies of SCEs in the first mitosis after their administration. The experiments with PHA-stimulated lymphocytes in vivo from patients treated with cytostatics showed that cytosine arabinoside, in combination with thioguanine, did not induce higher frequencies of SCEs, whereas in patients who were treated with cyclophosphamide alone or in combination with other cytostatic drugs, there was a higher incidence of SCEs during treatment. About 10 days after the termination of the treatment the elevated freuqencies of SCEs returned to the initial level. After administration of some mutagens, especially alkylating agents in vivo, the lymphocyte system can be used to assess induced DNA repair by continuously monitoring for SCEs.     

Toxicity of novel anthracycline derivatives towards normal myeloid bone marrow progenitor cells (CFU-GM) is not increased by verapamil

Drug-induced myelotoxicity is usually the dose-limiting factor of treatment of malignant tumors with cytostatic drugs. Suppression of in vitro myelopoiesis (CFU-GM) by cytostatics may be a suitable model reflecting the in vivo situation. Thus the inhibitory effects of the anthracyclines doxorubicin, theprubicin, idarubicin and cytorhodin S on CFU-GM were compared. Normal human bone marrow cells were incubated with these drugs for one hour and alternatively, for the whole culture period. For each substance and each incubation time a dose-response curve was established and the D50 determined. As certain calcium antagonists can increase the toxicity of some cytostatic drugs in various tumor models, the effect of the addition of verapamil (2 microM) was also investigated. It could be shown, that the myelotoxicity on CFU-GM of the drugs mentioned above was not increased after short-term or permanent exposure to this calcium antagonist.     

Studies on the effect of various kinds of cytostatic therapy on the cellular immune reaction in children with acute lymphatic leukemia]

Clinical and experimental findings on possible changes of the lymphocyte function during an immunosuppressive or cytostatic therapy respectively caused investigations to be made for explaining the connections existing between the influence of cellular immunoreaction and the use of different cytostatic regimes. Earlier findings on the influence of cellular immunoreaction after adding cytostatics to cultivated cells and investigations on the influence of the lymphocyte function in dependence on cytostatic therapy were used for comparison. Transformation and mitosis rates as well as necrosis rates and the result of macrophage migration inhibition are comparable parameters for influencing the lymphocyte function in children treated with cytostatics. Antimetabolites, vincristine, asparaginase and daunomycin will have less influence on the transformation rate as an expression of an immunosuppressive effect on only those cells responding in accordance with their kinetic phase. Cyclophosphamide will inhibit the transformation reaction more significantly. Examinations in children with different therapeutic regimes reveal a certain validity of therapy after the first statistical evaluation of the clinical material.     

Inhibition of normal granulopoiesis by cytostatic agents.

Rabbits were treated with cyclophosphamide and 5-fluorouracil, myelosuppressive cytostatic agents, applied with a single dose of 1/3 LD50 or daily doses of 1/30 LD50 given for 14 days. Functional tests for evaluation of granulopoiesis were regularly performed at standard intervals and were following: leukocytosis, bone marrow picture with mitotic index, 3H-thymidine incorporation in vitro followed by autoradiography of labeled promyelocytes and myelocytes, serum lysozyme activity, mobilization of granulocyte reserve pool by staphylococcal alpha-toxin, cytochemistry of granulocytes, phagocytosis ability and Nitro-BT reduction. It has been found that 6-10 days after application of cytostatics, a marked depression of proliferation of young granulocyte forms and lowered reserve pool, are regularly observed. This was followed by spontaneous regeneration of granulopoiesis. No changes were noted in functional tests of mature granulocytes in peripheral blood. It is suggested that for investigation of the impairment of granulopoiesis after application of cytostatic agents, most suitable is evaluation of mobilization of the bone marrow reserve pool, lysozyme activity in blood serum and labelling of promyelocytes and myelocytes with 3H-thymidine in vitro.     

Effects of modulators and cytostatics on catabolism of I-125 desoxyuridine in tumors (express-method of antineoplastic modulator screening)]

El-4 and P-815 murine tumor cells labelled by 125I-deoxyuridine or 51Cr were administered in 7-day subcutaneous syngeneic tumors or subcutaneously. At the same time different groups of mice were treated by LPS plus MDP, beta-C7H15-MDP, dexal-MDP, polyacrylamide-MDP-phosphatidylethanolamine, adriblastin or cyclophosphamide. It was shown that cytostatics and immunomodulators significantly delayed catabolism and withdrawing of 125I-deoxyuridine (that has not been incorporated in DNA) from tumor cells. This delay was correlated with the inhibition of tumor nodes growth rate. It is concluded that influence of cytostatics and immunomodulators on catabolism and withdrawing rate of 125I-deoxyuridine from tumor cells relates to their cytostatic effect and may be used at the earliest screening step of immunomodulator analysis.     

In vitro proliferation of normal and leukaemic human leukocytes controlled by an inhibitory endopeptide

GI-3, an endogenous inhibitory fraction isolated from leukocytes, selectively inhibits the proliferation of granuloid precursor cells in a non-toxic manner. Its active principle was determined as an acidic chlor-tolidine positive decapeptide [ 3 ]. The in vitro effect on normal and acute leukaemic human bone marrow and blood cells was examined. A dose dependent inhibition by GI-3 of 3H-TdR incorporation into myeloid cells of normal bone marrow was found, the sensitivity of human cells being higher than that of rat cells. The proliferation of the target leukaemic bone marrow and blood cells (AML, AMMoL) was also decreased by the endogenous inhibitor in a dose dependent manner in untreated subjects as well as in patients in remission or relapse. The rate of inhibition of leukaemic of well-known cytostatics (adriamycin hydrochloride, dianhydrogalactitol) applied for comparison. Beyond its direct cytostatic effect, GI-3 could be used in the differential diagnosis of blastic leukaemias, complementing the routine cytochemical methods.     

Analysis of changes in natural cytostatic and cytotoxic activity of mouse spleen cells after treatment with cyclophosphamide and alpha-interferon

Cytostatic and cytotoxic activity of mouse spleen cells against normal and tumour target cells has been studied. The comparative analysis of mouse spleen cell cytostatic and cytotoxic activity after exposure to cyclophosphamide has shown that the effectors of natural cytotoxic activity are highly sensitive to cyclophosphamide, while cytostatic effectors are heterogeneous in their sensitivity to cyclophosphamide. Pretreatment of spleen cells with alpha-interferon produced an increase in cytotoxic and cytostatic activity against tumour target cells. The cells of lymphoid organs (spleen, bone marrow, thymus) had greater distinctions in cytotoxic than in cytostatic activity against tumour target cells.     

The effects of cyclophosphamide on in vitro cytotoxic responses to a syngeneic tumour

Summary We have studied the effects of treating DBA/2 mice with high doses of cyclophosphamide upon their subsequent ability to generate cytotoxic cells in vitro against syngeneic tumour antigens or alloantigens. High doses of cyclophosphamide (100–200 mg/kg body weight) eliminated the response to both antigens. The addition of normal DBA/2 thymocytes into these cultures restored the response to allogeneic cells but not to tumour cells. The anti-tumour response could be restored by the addition of interleukin 2 to the cultures. Treatment with high doses of cyclophosphamide decreased the number of anti-tumour cytotoxic cell precursors in the spleen, but did not affect the capacity of bulk cultures of spleen cells to produce interleukin 2 when stimulated with the mitogen concanavalin A.Abbreviations CY Cyclophosphamide - CTL cytotoxic T cells - CTLp precursor cytotoxic T cells - IL2 interleukin 2 - Con A concanavalin A - FCS fetal calf serum     

The effect of therapy with cytostatics and immunomodulators on mitotic pathology in Lewis tumor cells in mice]

The effect of cytostatics (methylnitrosourea and methotrexate), immunomodulators (thymalin and reaferon) and their combinations on the mitotic pathology of mice Lewis tumour cells was studied. It was revealed that chemotherapy with these agents changed the interrelation between mitotic phases and somewhat enhanced the incidence of pathologic mitoses mainly connected with the damage of mitotic apparatus. Immunomodulators differently affected the cytostatic activity of antitumour agents that may be associated with their mechanisms of action.     

Mutagenicity of cytostatic drugs in a bacterial system

A set of nine cytostatic drugs were tested in the standard plate incorporation method of Ames reversion test using fourSalmonella typhimurium his strains. Six of them were classical and commonly used cytostatics, three (cloturin, butocin and oracin) are at different stages of development. The results showed 6-mercaptopurine, cloturin, adriamycin, mitoxantron, cyclophosphamide and lomustine to be mutagenic; butocin, oracin and tris(2-chloroethyl)amine were negative in this test.     

Comparative Investigations On the Effect of Different Dose Schedules of the Phase-Specific Drug Vincristine (Vcr) On the Proliferation Kinetics of A Solid Experimental Tumour

The aim of the present investigation was to study the effect of a high bolus injection (1 × 2.1 mg) of vincristine (VCR) during the phase of tumour growth retardation at the 14th day after transplantation and to compare the findings with the results of single (1 × 0.35 mg) and repeated (6 × 0.35 mg) applications of this cytostatic drug. Furthermore, an attempt was made to induce a synchronization of tumour cell proliferation. It was found that the effect on the volume growth was very pronounced after the high bolus injection and the repeated application of vincristine compared with the single low dose of the cytostatic drug. A synchronization of the tumour cell proliferation by flow out of the mitotic block could not be demonstrated. On the other hand a modest simultaneous recruitment of previously non-cycling tumour cells into the cell cycle occurred in the periphery of the tumour after the high bolus injection. the repeated application and the high bolus injection of VCR increased the cytostatic effect, especially in the tumour centre, related to the more slowly proliferating tumour cell compartment.     

Comparative investigations on the effect of different dose schedules of the phase-specific drug vincristine (VCR) on the proliferation kinetics of a solid experimental tumour

The aim of the present investigation was to study the effect of a high bolus injection (1 X 2.1 mg) of vincristine (VCR) during the phase of tumour growth retardation at the 14th day after transplantation and to compare the findings with the results of single (1 X 0.35 mg) and repeated (6 X 0.35 mg) applications of this cytostatic drug. Furthermore, an attempt was made to induce a synchronization of tumour cell proliferation. It was found that the effect on the volume growth was very pronounced after the high bolus injection and the repeated application of vincristine compared with the single low dose of the cytostatic drug. A synchronization of the tumour cell proliferation by flow out of the mitotic block could not be demonstrated. On the other hand a modest simultaneous recruitment of previously non-cycling tumour cells into the cell cycle occurred in the periphery of the tumour after the high bolus injection. The repeated application and the high bolus injection of VCR increased the cytostatic effect, especially in the tumour centre, related to the more slowly proliferating tumour cell compartment.     

Augmentation of host antitumor immunity by low doses of cyclophosphamide and mafosfamide in two animal tumor models

Summary By cloning in vitro we have obtained two sublines of the L5222 rat leukemia, one with high (L5222-S) and the other with low (L5222-R) in vivo sensitivities to non-toxic doses of mafosfamide, a stabilized derivative of 4-hydroxy-cyclophosphamide. This sensitivity in vivo was not related to the cytotoxic activity of the drug in vitro. Treatment of rats bearing the L5222-S and of mice transplanted with the MOPC-315 plasmocytoma with low doses of mafosfamide or cyclophosphamide resulted in a high percentage of surviving animals, which were resistant to a subsequent tumor challenge. Viable leukemic cells were needed to establish antitumor immunity, since it was not possible to induce resistance by injection of mitomycin-C-treated, non-viable L5222 cells. The adoptive transfer of spleen cells from animals immune against the L5222-S and the MOPC-315 resulted in resistance of the syngeneic recipients against a rechallenge with tumor cells, provided that the animals were treated with an immunosuppressive dose (100 mg/kg) of cyclophosphamide prior to the spleen cell implantation. In nude mice treatment of the L5222 with low doses of mafosfamide also resulted in surviving animals, however resistance to a second tumor challenge occurred only sporadically.The data presented confirm that therapy with cyclophosphamide or mafosfamide enhances host antitumor immunity but, contrary to previous reports, it could be demonstrated that successful tumor rejection was independent of T cells.Supported by the Federal Ministry of Research and Technology (BMFT), Bonn-Bad Godesberg, FRG     

Cyclophosphamide and abrogation of tumor-induced suppressor T cell activity

Summary Previously we have demonstrated that the in vitro generation of P815-specific anti-tumor cytotoxic T lymphocytes (CTL) was suppressed by splenic suppressor T cells from late tumor-bearing hosts (TBH). Suppression is not caused by in vitro growth of P815 from splenic metastases, since suppression was also seen with spleen cells from late TBH mice bearing a hypoxanthine/aminopterin/thymidine-sensitive subline (PHS-5) of P815 in the presence of HAT. Cyclophosphamide has been shown to inhibit theinduction of suppressor cells selectively in a number of immune responses, but evidence that it can inhibit active tumor-induced suppressor T cells is limited. We have found that suppressor T cells already induced by P815 in syngeneic late TBH are sensitive to low doses of cyclophosphamide (50 mg/kg) given 1 day before spleen harvest, but the in vitro CTL response of late TBH spleen cells could not be restored by pretreating the mice with cyclophosphamide, even when exogenous interleukin-2 was added to the cultures. Although 50 mg/kg cyclophosphamide did not inhibit the CTL response of spleen cells from mice immunized with P815 +Corynebacterium parvum, the same dose of cyclophosphamide eliminated the CTL response of spleen cells from early TBH. Interleukin-2 (IL-2) did not overcome this effect of cyclophosphamide, suggesting a direct effect on CTL. Ultra-low-dose cyclophosphamide (10 mg/kg) did not adversely effect early TBH CTL but was still able to eliminate suppressor T cell activity from late TBH. Nevertheless, late TBH CTL remained unresponsive after pretreatment of mice with ultra-low-dose cyclophosphamide, even when exogenous IL-2 was added in vitro. CTL precursor frequency analyses demonstrated that cyclophosphamide pretreatment had little or no effect on the numbers of CTL precursors from early TBH. Late TBH CTL precursor cells were not detectable in these studies, with or without suppressor T cell inhibition by cyclophosphamide pretreatment. Thus, it appears that most CTL precursor cells may be lost or irretrievably inactivated in the spleens of late TBH mice.This work was supported by grants CA42443, CA48075 and T32-CA09210 from the National Cancer Institute, Department of Health and Human Services, and an American Cancer Society Clinical Oncology Career Development Award (H. D. Bear)     

Induction of the tumoricidal activity of human and murine peritoneal macrophages under the action of antitumor chemical preparations

Platidiam, cyclophosphamide and adriamycin induced tumoricidal activity of peritoneal macrophages from patients with disseminated ovarian carcinoma when applied in the autologous tumor cells in vitro. This effect was not observed with 10 micrograms/ml concentration of 5-fluorouracil. The mice peritoneal macrophages after incubation in vitro with 0.01-1.0 micrograms/ml of aclarubicin showed cytostatic action on syngeneic and semisyngeneic P388 cells. The peritoneal macrophages from mice treated with 2.5 mu/kg of aclarubicin intraperitoneally 1-4 days before were cytotoxic for tumor cells too.     

Cultured Nb rat lymphoma cells in endocrine and cancer research

This review outlines the establishment, properties, and use of two lines of cultured Nb rat lymphoma cells. The cultured cells have retained important properties of the cancers of origin, such as dependency on prolactin for growth and a high sensitivity to antineoplastic Vinca alkaloids. The cultures have been useful for defining the hormonal dependency of the lymphomas in the animal and for studying the progression of the lymphomas from hormonal dependency to autonomy. A new, specific and highly sensitive in vitro bioassay for lactogenic hormones has been developed from one of the cultures. The use of the lymphoma cell cultures has revealed unsuspected pharmacological differences between the closely related, clinically useful antineoplastic Vinca alkaloids, vinblastine and vincristine. The lymphoma cell cultures are also useful tools for studying biochemical, cell cycle related events which follow the mitogenic stimulation of cells by a polypeptide growth factor.     

The effect of different infectious agents on the intensification of hematopoiesis during immunosuppression

The action of Staphylococcus aureus, Escherichia coli and Herpes simplex virus on the hemopoiesis of mice with cytostatic myelosuppression was studied. The study revealed that the infection of the animals simultaneously with the action of cyclophosphamide considerably activated the processes leading to the restoration of hemopoiesis due to an increase in the mitotic activity of hemopoietic cells, the accelerated differentiation of hemopoietic precursor cells which could survive the cytostatic action and an increase in the functional activity of the hemopoiesis-producing microenvironment.     

The use of cytotoxic T cells in the regulation of tumour growth in syngeneic mice

Summary Normal C57BL/6 (B6) spleen cells were cultured with syngeneic EL4 tumour cells, expanded in IL2-containing medium, and tested for anti-tumour activity in vitro and in vivo. The activated cells were highly cytotoxic for EL4 and to a lesser degree killed syngeneic B6 blasts and allogeneic (D2) P815 tumour cells. B6 or BDF1 mice that received these cultured cells by IP injection cleared ¹²⁵IUdR-labelled EL4 cells faster than untreated mice. However, this enhanced clearance was evident only 7–12 days after injection. Since the injected cells had a short half-life (<10% remaining after 48 h) the effect of these cells in vivo was most probably due to the activation of the host's immune system. Mice that received cultured cells survived significantly longer than untreated mice following a lethal dose of EL4 cells. Cultured cells were much more effective in prolonging survival when used in conjunction with cyclophosphamide (CY). In animals receiving either cultured cells with or without CY or CY alone tumour clearance was markedly enhanced 7–12 days after injection.When challenged with a small dose of EL4 tumour cells (1×10⁴ SC per mouse) three of ten B6 mice treated with B6 anti-EL4 cultured cells were able to survive indefinitely. The frequency of CTL precursors to EL4 from the spleen cells of these surviving animals was about five-fold higher than that of normal spleen cells. Furthermore, CTL derived from primed spleen cells were more specific for EL4 than those derived from normal spleen cells.Abbreviations B6 C57BL/6J - BDF1 (C57BL/6J×DBA/2J) F1 - ConA SN concanavalin A supernatant - CTL cytotoxic T lymphocytes - CTL-P cytotoxic T-lymphocyte precursors - CY cyclophosphamide - E/T effector-to-target ratio - IL2 interleukin 2 - IP intraperitoneal - IUdR iododeoxyuridine - IV intravenous - LPS lipopolysaccharide - MST mean survival time     

Differential effects of drugs upon hematopoiesis can be assessed in long-term bone marrow cultures established on nylon screens.

Hematotoxicity is associated with exposure to chemotherapeutic drugs and numerous other agents. Most measurements of the hematopoietic effects of prospective therapeutic drugs and environmental agents have been made in animal models. We tested the influence of various drugs on hematopoiesis in long-term cultures of Long-Evans rat bone marrow cells. These cultures were established on nylon screen-bone marrow stromal cell templates that were suspended in liquid medium. Previous phenotypic analyses of adherent zone cells of suspended nylon screen bone marrow cultures (NSBMC) using monoclonal antibodies and flow cytometry indicated that they maintain a multilineage character for extended periods in culture and display continuous proliferation of hematopoietic progenitors (colony-forming unit culture [CFU-C]). NSBMC of various ages were incubated for 21 hr with several concentrations of beta-D-cytosine arabinofuranoside, 5-fluorouracil, cyclophosphamide, or methotrexate. Adherent zone cells were dissociated enzymatically, phenotyped by flow cytometry, and assayed for colony-forming unit culture content. beta-D-cytosine arabinofuranoside, 5-fluorouracil, and methotrexate treatment of bone marrow cultures resulted in a dose-related diminution in colony-forming unit culture numbers in the adherent zones of NSBMC. Phenotypic analyses revealed similar trends but certain of these drugs manifested lineage specificities. Toxicity was also related to cyclophosphamide dose, but the presence of bone marrow stroma was necessary to demonstrate this effect in vitro. A subpopulation of these cells was found to metabolize ethoxyfluorescein ethyl ester to fluorescein after induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin, an effect which was quantified by flow cytometry. NSBMC may be used to ascertain lineage-specific toxicities and evaluate the effects of drugs on the proliferation of hematopoietic progenitor cells.