Spleen Colony Assay Research Articles

Enhancing effect of misonidazole on the response of the RIF-1 tumour to cyclophosphamide.

The effect of misonidazole (MISO) on the cytotoxicity of cyclophosphamide (CY) was investigated in the mouse. The response of the RIF-1 tumour was measured by growth delay and by cell survival in a cloning assay. MISO enhanced the cytotoxicity of CY. For single treatment, enhancement was maximal when MISO was given 30 min to 2 h before CY. The enhancement ratio (i.e. the dose of CY alone divided by the dose of CY with MISO required to cause the same response) increased with increasing dose of MISO up to 250 mg/kg, but decreased with increasing dose of CY above 50 mg/kg. For 5 daily treatments, enhancement increased with CY dose up to approximately 25 mg/kg/injection. Survival of marrow stem cells was measured using the spleen-colony assay. MISO did not enhance significantly the cytotoxicity of CY at doses under 100 mg/kg. Enhancement was seen at higher doses, but the effect was less than in tumours. CY reduced the number of circulating white blood cells. Neutrophils were most severely depleted. The WBC count was slightly lower when CY was given in combination with MISO than after CY alone, but the effect could be accounted for by direct MISO cytotoxicity. These experiences suggest that a therapeutic gain may be achieved if MISO is combined with doses of CY in the clinical range. From experiments performed to investigated the possible mechanisms involved, we conclude that for the RIF-1 tumour the major effect of MISO is to inhibit the repair from CY-induced potentially lethal damage.

Potentiation of Anticancer Agents by Amphotericin B<xref ref-type="fn" rid="FN2">2</xref>

Amphotericin B (AmB) has been shown to potentiate the cytotoxicity of several different anticancer agents in AKR mice. The extent of potentiation, as assessed quantitatively by a spleen colony assay, varied from a factor of approximately 3 for 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU) to over 100 for a number of alkylating agents and to over 1,000 for 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Most phase-specific agents showed little potentiation of cytotoxicity by AmB. A dose-response effect was noted with both BCNU and adriamycin, with increased potentiation being observed with increasing doses of the anticancer agents. The effect on increase in life-span was also studied and for most of the combinations it correlated with the potentiating effects disclosed by the spleen colony assay. Such a correlation was not found for AmB combined with either BCNU or vincristine.

Cytotoxic effect of 5-fluorouracil plus cyclophosphamide against transplantable leukemias

The cytotoxic effect of the combination of cyclophosphamide and 5-fluorouracil against AKR and L1210 leukemias was quantitated by a spleen colony assay. We used different sequences, a number of doses of each agent, and different intervals between the agents and noted different degrees of synergistic cell-kill. By proper scheduling of these agents, greater than 100-fold increase in cell killing was noted, an effect not demonstrable for normal hematopoietic stem cells. However, the pattern of response for AKR was opposite to that for L1210 leukemia; we suggest that this reflects a difference in the metabolism of 5-FU.

Utilization of mouse stem cell-depleted marrow in the study of diffusion chamber myelopoiesis.

AbstractMouse brain antisera with anti-CFU-S activity was utilized to evaluate the role of the pluripotent stem cell (CFU-S) in a diffusion chamber (DC) myelopoiesis. Antisera incubation with marrow resulted in a 93 ± 2% reduction in detectable CFU-S in the standard spleen colony assay and a variable reduction in the granulocyte-monocyte progenitor cell (CFU-C). Marrow virtually devoid of detectable CFU-S and containing variable CFU-C relative to control demonstrated equal or greater numbers of CFU-S, CFU-C, and differentiated progeny at varying time intervals after DC implantation. These results may indicate that neither the CFU-S nor the CFU-C is the progenitor cell for DC growth. Alternatively, CFU-S antibody may not kill CFU-S in vitro or in the in vivo DC system but may opsonize the cell prior to eventual reticuloendothelial destruction. The latter concept is supported by reversal of antisera effect on CFU-S recovery after in vitro exposure of antisera-treated cells to pronase.

Growth Characteristics of MOPC-315 Plasmacytoma and Response to Anticancer Agents2, 3

We examined the growth characteristics and response to anticancer agents of the murine plasmacytoma MOPC-315, a rapidly growing and widely disseminating tumor syngeneic in BALB/c mice. The doubling time for the tumor was approximately 24 hours, and about 1 in 100 cells was tumorigenic. We developed a spleen colony assay for the clonogenic component of the tumor and used it to define the dose-response relationships for various anticancer agents, including melphalan, cyclophosphamide, 1,3-bis(chloroethyl)-1-nitrosourea, 5-fluouracil, gamma-radiation, methotrexate, methylprednisolone, cytosine arabinoside, and vincristine.

The proliferative state of normal hemopoietic stem cells during progression of leukemia. Studies in the BN acute myelocytic leukemia (BNML)

The kinetics of proliferation of normal hemopoietic stem cells (HSC) were investigated during the course of leukemia in a rat model for human acute myelocytic leukemia (BNML). The fraction of stem cells in DNA synthesis phase was determined with the hydroxyurea suicide technique followed by modified spleen colony assays on bone marrow, blood and spleen cell suspensions. It was found that, besides a redistribution and an absolute numerical decrease in the total number of HSC (to 45% of the original number), the percentage of cycling HSC decreased significantly as the leukemia progressed. In the bone marrow the S-phase fraction of HSC decreased from 38% to almost zero. Proliferating stem cells are thought to emigrate from the blood and lodge at extramedullary sites. Proliferation of HSC is best preserved in the spleen (in the terminal stage: 32 to 53% in S-phase). However, normal hemopoiesis is still insufficient and the rats die from the consequences of a lack of mature end cells. A possible mechanism underlying the changes and differences in kinetic behaviour of HSC in the various organs studied is discussed.

Suppression and Potentiation of Mouse Hematopoietic Progenitor Cell Proliferation by Ouabain

Clonal assays for CFU-S, CFU-C, BFU-E, and CFU-E were employed to evaluate the effect of ouabain on the proliferation of mouse hematopoietic progenitor cells. Preincubation of bone marrow cells with ouabain at concentrations of 10–6–10–12 M suppressed the proliferation of CFU-S as measured by the spleen colony assay. At 10–9M ouabain, spleen colony formation was inhibited by more than 95%. When included in soft agar cultures of bone marrow cells, ouabain suppressed the proliferation of CFU-C in a complex fashion. At 10–4M ouabain, colony formation was suppressed by 70%, while at 10–6M ouabain, CFU-C proliferation was normal. At 10–9M ouabain, however, the number of colonies formed was only 70% of normal, and complete recovery was not obtained at 10–12M ouabain. The number of colonies formed by BFU-E and CFU-E in plasma clot cultures was also diminished in the presence of 10–4M ouabain. In contrast to its effect on CFU-S and CFU-C, however, 10–9M ouabain enhanced BFU-E colony formation by 200% and CFU-E colony formation by 35%; at 10–12M ouabain, CFU-E proliferation was unaffected by the drug. These data indicate that mouse CFU-S, CFU-C, BFU-E, and CFU-E require a functional membrane-bound Na+K+ATPase for proliferation; that these progenitor cells differ in their response to ouabain and finally, that ouabain at concentrations below that (10–6M) considered necessary to inhibit membrane-bound Na+K+ ATPase can influence the proliferation of mouse hematopoietic progenitor cells.

Therapeutic Synergism of Hyperthermia-cis-Platinum in a Mouse Tumor Model23

A small-animal model was developed as a guide to whole-body hyperthermia in cancer patiens. Anesthetized DBA/2 mice were secured to a platform, and their hindlimbs were immersed in a 42.3 degrees C water bath for 30-60 minutes. Hindlimb hyperthermia reulted in steady-state rectal and femoral bone marrow and muscle temperatures of 42 degrees C and upper extremity muscle and esophagus temperatures of 41 degrees C. With this hyper thermia technique, the mouse spleen colony assay could be used to quantitate the lethality of hyperthermia and/or cis-dichloro-diammineplatinum(II) (cis-platinum) on clonogenic bone marrow and leukemia cells. Hyperthermia prior to cis-platinum administration increased cis-platinum inhibition of leukemia colony formation as much as 2 logs; however, antileukemia synergism ws greatest when cis-platinum administration immediately preceded hyperthermia and no evidence existd of synergism against normal bone marrow colonies. Correlative in vivo drug uptake studies showed a marked increase in leukemia cell uptake of 195mPt-cis-platinum at elevated temperatures, which suggested a potential mechanism for the apparent antileukemia synergism of cis-platinum and heat.

Cytotoxicity of Adriamycin Combined With Methotrexate Against L1210 Leukemia in Mice<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

The combination of adriamycin and methotrexate was found in mice to be synergistic in cytotoxicity to L1210 leukemia cells for a range of time intervals between the two agents. Cytotoxicity for normal hematopoietic stem cells was less than additive. Spleen colony assays were used to quantitate both cell populations. Increase in life-span assays yielded similar results. Also, the extent of synergy was dependent on the dose of adriamycin only.

In Vivo Potentiation of 5-Fluorouracil Cytotoxicity Against AKR Leukemia by Purines, Pyrimidines, and Their Nucleosides and Deoxynucleosides<xref ref-type="fn" rid="FN1">2</xref>

The effect of various natural pyrimidines and purines and their nucleosides and deoxynucleosides on 5-fluorouracil (FUra) cytotoxicity was examined against the transplantable AKR leukemia in female AKR mice. The spleen colony assay was used for a quantitative evaluation of the antitumor activity of the combination treatments. The base or nucleoside was administered 15 minutes before each mouse received an injection of 0.6 mg FUra. All of the compounds tested, with the exception of adenine, potentiated the cell-killing effect of FUra. On a molar basis (16-30 mumol/mouse), thymine, uracil, thymidine, and uridine enhanced FUra cytotoxicity more than a hundredfold. 2'-Deoxyuridine and the purine nucleosides and deoxynucleosides had similar potentiating activities between tenfold and fortyfold at 30 mumol per mouse. Finally, glucose, also administered 15 minutes before each mouse received an injection of 0.6 mg FUra, enhanced the antitumor activity of the drug by a factor of about five.

Hemopoietic activities of cryopreserved murine fresh and postmortem bone marrow cells

The percentage of preservation of erythropoietic and granulopoietic precursor cells in the murine bone marrow was studied using in vitro methylcellulose clonal cell culture assays and in vivo murine spleen colony assays. This study clearly demonstrates a Type of Spleen Colonies Induced by 6-hr Postmortem Murine Bone Marrow Cells a Mean (%) Type of colonies t Score P Value Unfrozen Frozen Erythrocytic 26.283 14.100 2.09 0.059 Granulocytic 23.741 32.917 1.45 0.173 Mixed 49.321 52.700 0.55 0.59 a N = 92. the presence of pluripotent hemopoietic precursor cells in cryopreserved 0-, 3-, 6-, 9-, and 12-hr postmortem murine bone marrow cells. Apparently, the erythropoietic precursor cells are more sensitive to freezing injury as compared to granulopoietic precursor cells.

Properties of haematopoietic stem cells surviving 5-fluorouracil treatment: evidence for a pre-CFU-S cell?

The spleen colony assay of Till and McCulloch1, which is a clonal assay2, is considered to be a measure of the stem cell content of haematopoietic tissue. Stem cells are a heterogeneous population3–6 with respect to their capacity to generate further stem cells (assayed as colony-forming units in spleen, CFU-S) and progenitors of red cells, granulocytes, macrophages and platelets. It has been suggested that stem cells are organised for use according to their generation age5,6. There is evidence to indicate that drugs such as hydroxyurea (HU) and 5-fluorouracil (5-FU), which preferentially eliminate proliferating cells, spare non-cycling stem cells which have a high capacity to generate other stem cells6 and progenitors of granulocytes and macrophages6 as well as megakaryocytes7. We show here, however, that the CFU-S content of haematopoietic tissue is not an adequate predictor of the capacity of that tissue to repopulate bone marrow of a lethally irradiated host. Furthermore it appears that haematopoietic stem cells are not only heterogeneous with respect to their capacity to generate other stem cells and progenitors of haemic cells in the spleen but also differ in their capacity to ‘home’ to either the bone marrow or spleen. The evidence suggests a class of ‘primitive’ haematopoietic stem cell (pre-CFU-S) that accumulates in the bone marrow to give rise to cells capable of growing in spleen.

Effects of whole body irradiation in mice treated with C. parvum

We have investigated the effect of whole body irradiation (WBI) upon the survival of Corynebacterium parvum ( C. parvum)-treated CBA mice. Applied to mice 7 days after C. parvum WBI with 350–950 rad of X-ray killed more C. parvum-treated than -untreated mice. The number of endogenous hematopoietic colonies determined either at 9 days of irradiation or at the time of death of mice was greater in C. parvum-treated mice than in mice irradiated only. Using the exogenous spleen colony assay, it was found that CFUs from the spleen of mice treated with C. parvum were more sensitive to irradiation than the CFUs from the spleen of normal mice, Leucopenia caused by irradiation was equally severe in both groups of mice. However, a decrease in the number of erythrocytes was more pronounced in mice treated with C parvum and irradiation than in those given irradiation alone. Possible reasons for the increased radiosensitivity of C. parvum-treated mice as well as the relevance of these findings to tumor immunotherapy are discussed.

THE RELATIONSHIP BETWEEN STEM CELL SEEDING EFFICIENCY AND POSITION IN CELL CYCLE

The seeding efficiency of colony-forming cells from normal, regenerating and velocity-sedimented cycling and non-cycling narrow preparations was compared. Colony-forming cells in cycle were found to exhibit a 50% reduction in splenic seeding when compared to normal marrow or sedimented non-cycling cells. The results of this study indicate that the spleen colony assay underestimates the total number of colony-forming cells by a fraction which is directly related to the number of cells in cycle.

Effect of ricin and abrin on survival of L1210 leukemic mice and on leukemic and normal bone-marrow cells.

The effect of ricin and abrin on the survival of mice treated with L1210 leukemic cells intraperitoneally or intravenously was studied. In mice given 1 X 10(5) L1210 leukemia cells intraperitoneally a single dose of ricin (2.1 microgram/kg) intraperitoneally gave the best results, an increased life span (ILS) of 59%. Abrin also increased the life span of such animals although to a lesser extent. The effect of ricin was superior to that of 5-fluorouracil, but inferior to that of adriamycin, which gave a maximum ILS of 280%. In mice given L1210 cells intravenously no increase in life span was obtained with ricin, abrin or adiramycin, whereas 5-fluorouracil gave an ILS of 40-50%. In spleen colony assays the differential effect of ricin and abrin on the proliferative capacity of normal hematopoietic and leukemic colony-forming cells in bone marrow was studied. The differential effect of ricin was as good as that of adriamycin and considerably better than that of 5-fluorouracil. Abrin had a much smaller effect than ricin on both normal and leukemic cells. The effect of abrin on the leukemic cells was too small to be of therapeutic value. The results warrant exploration of the use of ricin in the treatment of human leukemia.

In Vivo Enhancement of 5-Fluorouracil Cytotoxicity to AKR Leukemia Cells by Thymidine in Mice<xref ref-type="fn" rid="FN2">2</xref>

The spleen colony assay was used to examine the effect of thymidine (dThd) on 5-fluorouracil (FUra) cytotoxicity in two transplantable leukemias, AKR (in AKR mice) and L1210 [in (BALB/c x DBA/2)F1 mice], in vivo. A large dose of dThd (10 mg/mouse) could not rescue these cell lines from FUra toxicity. Instead, when dThd was given within 1 hour before FUra, it enhanced FUra cytotoxicity by a factor between 100 and 1,000 in AKR leukemia. That dThd increased the cytotoxicity of FUra only by a factor of 3 in L1210 leukemia suggested a different mechanism of interaction of the two drugs in the two cell lines. Examination in hybrid mice capable of supporting the growth of both leukemias showed the enhancement to be tumor related rather than host related. We also demonstrated a dose-dependent effect of dThd injection 15 minutes before FUra in AKR leukemia. Concerning the kinetics of killing of AKR leukemia colony-forming units (LCFU) following the administration of dThd 15 minutes before FUra, LCFU survival continued to decrease for 24--36 hours following drug administration.

HAEMOPOIETIC STEM CELLS IN THE FOETAL MOUSE THYMUS

The presence of haemopoietic stem cells (HSC) in the foetal mouse thymus was assessed to determine whether all cells which enter the developing organ are precommitted to thymocyte differentiation, or if stem cell multipotentiality still exists. The Till and McCulloch spleen colony assay was used to delineate foetal-thymus derived HSC in lethally irradiated recipients. Of the range examined, between 13 days of gestation to birth, a peak of stem cell activity occurs in 15-day foetal thymus. The surface colonies produced by the thymus-derived HSC are small compared to colonies produced by the liver derived HSC, although well within the range of the latter. Histologically, five types of colonies were identifiable which were produced by the thymus-derived HSC, indicating that these cells retain the potential to form a wide range of differentiated colonies.

A target cell assay for friend virus

A method has been developed for the in vitro infection and subsequent quantification of murine hemopoietic target cells with FV grown in vivo. The number of infected FV releasing cells is quantified in a spleen colony assay for infectious centers. The hemopoietic nature of these cells is demonstrated by the fact that bleeding increases the number of target cells in spleen and bone marrow of mice. This procedure may be of significance in the study of both FV-target cell interactions and the problem of oncogenic transformation by FV.

Tumor-related blocking of anti-fetal immunity

A modified spleen colony assay was employed to evaluate the effect of various tumor-related factors upon the in vivo effectiveness of anti-fetal immunity. Reduction of fetal liver (hemopoietic) colony formation by in vitro incubation of fetal liver cells with lymph node cells sensitized to syngeneic fetal liver or plasma cell tumor was blocked by (1) solubilized fetal antigen, (2) serum from mice recently immunized with syngeneic fetal liver and (3) serum from patients with metastatic colon cancer. In the latter, the degree of blocking correlated with plasma CEA levels. Both humoral and cell-mediated mechanisms of anti-fetal immunity were blocked in plasma cell tumor-bearing mice, suggesting in vivo suppression of the immune response via circulating tumor-associated fetal antigen.

REGULATION OF BONE MARROW CELL GROWTH IN DIFFUSION CHAMBERS: THE EFFECT OF GRANULOCYTE EXTRACTS

Hysotonic lysis of mature human blood granulocytes yielded an extract which reduced granulopoiesis and enhanced macrophage formation of mouse bone marrow cells cultured for 7 days in diffusion chambers (DC). The low molecular weight fraction (MW less than 15,000-25,000 Daltons) obtained by Amicon filtration of the extract, reduced granulopoiesis without affecting macrophage formation. The high molecular weight fraction (MW greater than 15,000-25,000 Daltons) reduced the number of granulocytes and increased the number macrophages. Erythrocyte extract increased the macrophage formation in DC but did not alter the number of granulocytes. The spleen colony assay showed that the granulocyte extract increased the number of CFU-S in DC. It is suggested that the granulocyte extract contain an inhibitor of stem cell differentiation to myeloid cells thereby reducing the number of proliferative granulocytes in DC 7 days later. The inhibitor of differentiation may lead to an increased self renewal of the stem cell in the DC system.