Rat Leukemia Research Articles

High-dose cyclophosphamide treatment of acute myelocytic leukemia. Studies in the BNML rat model

In view of its application in patients with acute leukemia prior to bone marrow transplantation, the toxicity and efficacy of high-dose cyclophosphamide treatment were evaluated in a rat model for human acute myelocytic leukemia (BNML). The ld 50 in leukemic rats proved to be lower than that in normal rats (100 vs 164 mg/kg respectively). With dosages above 120 mg/kg, bone marrow transplantation was required to overcome irreversible aplasia. Additional causes of death were lung-, bladder- and intestinal tract hemorrhages (160–200 mg/kg) or acute cardiopulmonary failure (250–300 mg/kg). In leukemic rats, excessive leukemic cell kill leading to tumor cell embolism was another contributing factor. In this respect, treatment of late-stage leukemia proved invariably fatal. In leukemic rats, the highest therapeutic index was achieved with 100 mg/kg. Depending on the stage of disease, a 5–8.5 log leukemic cell kill was achieved. An increased proliferation rate of residual leukemic cells after cyclophosphamide treatment appeared likely. Finally, the present data are extrapolated to the current treatment of human acute myelocytic leukemia in complete remission with high-dose cyclophosphamide in combination with supralethal total-body irradiation.

Selective killing of malignant cells in a leukaemic rat bone marrow using an antibody–ricin conjugate

Selective killing of malignant cells in a leukaemic rat bone marrow using an antibody–ricin conjugate

Induction of fibrosarcoma by rat sarcoma virus

The rat sarcoma virus (RaSV) causes rapid transformation of rat embryo fibroblasts in vitro and induces a 29,000-dalton protein (pp29) in the transformed cells. Several strains of newborn rats were inoculated with RaSV with its natural helper rat leukemia virus (RaLV) or RaSV pseudotypes of replication competent murine leukemia virus or baboon endogenous virus. Approximately 2–3% of susceptible rats developed fibrosarcomas in 12–20 weeks. However, none showed any immune response or viremia over a 10-month follow-up period. Further, we isolated RaSV from the in vivo induced tumors that transformed rat cells in vitro. Both the tumor and in vitro transformed cells exhibited the same gene product (pp29) as described earlier.

A recommendation for high-dose Ara-C interval treatment based on studies in a slow-growing leukemia model (BNML).

The arabinoside cytosine (Ara-C) dose-response relationship was investigated in a slow-growing rat leukemia (BNML) which is a realistic model for human acute myelocytic leukemia (AML). For both the tumor load reduction determined as leukemic colony-forming units spleen (LCFU-s) and the normal stem cell toxicity determined as colony forming units spleen (CFU-s), a plateau curve was observed. Maximum LCFU-s reduction was observed after high-dose Ara-C (200 mg/kg), while the greatest CFU-s reduction occurred following a ten fold lower dose. For the high Ara-C dose, several methods of administration were compared: rapid intravenous (IV), intramuscular, subcutaneous, intraperitoneal, and continuous infusion. The most effective tumor load reduction was obtained by rapid IV interval treatment. In addition, rapid IV interval administration of Ara-C was less toxic to normal bone marrow stem cells than continuous infusion of the drug. The results of these studies may justify the application of high-dose Ara-C interval treatment in human AML.

Extracorporeal Irradiation of the Blood in a Rat Model for Human Acute Myelocytic Leukemia. Increased Efficacy after Combination with Cell Mobilization by Low-Molecular-Weight Dextran Sulfate

The efficacy of extracorporeal irradiation of the blood (ECIB) in combination with cell mobilization by dextran sulfate (DS; MW 17,000) was investigated in a rat model for human acute myelocytic leukemia. Repeated injections with DS (q 3 hr) induced a significant increase in the number of peripheral leukemic cells, i.e., up to 4.5 times the original number 6 hr after the first injection. Cell mobilization in combination with ECIB (2 x 8 hr) caused a depletion of the blood compartments and the rapidly exchangeable tissue pool down to 10-25% of their original sizes, as determined by measuring the distribution of infused 51Cr-labeled leukemic cells and organ weights. These size reductions are about two times as great as those in rats treated with ECIB alone. In addition, the slowly exchangeable tissue pools are significantly depleted when DS is added to ECIB treatment. The reduction in the total tumor load was about 50%. This, however, is too small to result in a significant difference in survival time between treated and nontreated leukemic rats. Chemicals/CAS: chromium 51, 14392-02-0; dextran sulfate, 9011-18-1, 9042-14-2; Dextran Sulfate, 9042-14-2; Dextrans, 9004-54-0

Chemotherapy of autochthonous myeloid leukemias (Chloroleukemias) in Wistar rats.

After injection of 15 mg/kg ethylnitrosourea (ENU) weekly for 15 weeks to adult male Wistar rats (total dose: 225 mg/kg) about 10% of the animals developed myeloid leukemias (chloroleukemias), which resemble the chronic myeloid leukemia in man (CML) (peripheral blood picture, tissue infiltration, chronic course as compared to immature-cell rat leukemias). Monotherapy with busulfan effected no remissions. The median survival time after daily treatment with busulfan was 29.5 days (range: 7-70); it was significantly shorter than that of untreated controls (median: 47.5 days, range: 22-81). After weekly application of 20 mg/kg and 50 mg/kg Cyclophosphamide the median survival time increased to 69.5 (range: 26-114) and 61.5 (range: 20-92) days, respectively. Rate and duration of remissions after repeated weekly single doses of cyclophosphamide were positively correlated with the increase in single dose; the high dose-intermittent treatment with 50 mg/kg CPA/week yielded complete remissions in all treated animals. Despite these remissions however, no significant increase in survival time could be observed in comparison with untreated controls. The comparability of autochthonous chloroleukemias in the rat with human CML is discussed.

Chemotherapy of Leukemia in Mice, Rats, and Humans Relating Time of Humoral Stimulation, Tumor Growth, and Clinical Response<xref ref-type="fn" rid="fn2">2</xref><xref ref-type="fn" rid="fn3">3</xref>

Studies were conducted in the leukemic mouse and rat to test the hypothesis that enhanced effects of drugs given in sequence relate to a predictable increase in tumor growth and sensitivity to cycle-active agents. The rationale is based on a) evidence that, following drug-induced aplasia, resultant bone marrow proliferation in vivo corresponds temporally with induced humoral stimulatory activity, and on b) models that demonstrate increased cytotoxicity of beta-cytosine arabinoside (Ara-C) to myeloblasts cultured in humoral stimulatory activity (HSA). CD2F1 mice bearing L-1210 leukemia received a course of 60 mg Ara-C/kg every 8th hour (q.8 h) three times on day 0 and on another day in sequence (0,1 through 0,7). The longest survival (250% of controls) was in animals whose second course began on day 3, the time of peak HSA as measured by DNA synthesis induced in L-1210 cells in culture. LBN rats bearing acute myelocytic leukemia (AML) were treated with 100 mg Ara-C/kg q.8 h six times beginning on day 0 and on other days in sequence (0,1 through 0,12). The longest survival was in those treated on day 0,6 (760% of controls), the time of peak serum stimulation, and tumor labeling index (LI). Thirty-seven patients with AML received a single course of therapy with 45 mg Ara-C/kg by a 72-hour infusion and 1.0 mg daunorubicin/kg every day three times. On day 8, the time of peak HSA and tumor LI, Ara-C was again infused. Complete remission was achieved in 56% (65% of all patients less than 60 yr old) with a single cycle of therapy. Median duration of chemotherapy-free remission was 10 months. Of 11 relapsing patients, 8 achieved a second remission with the same regimen. These studies demonstrated that the amount of proliferation of residual tumor and thereby sensitivity to cycle-active drugs given in sequence relates to the initial drug effect on tumor proliferation and the induction of humoral stimulation.

Different distribution of adriamycin in normal and leukaemic rats.

Adriamycin (ADR) accumulates in well-perfused organs in the rat. This effect is especially evident for long periods in marrow and spleen of healthy animals. In rats bearing the Brown Norway Acute Myeloid Leukaemia (BNML) the in vivo distribution is significantly different. Maximum ADR levels in those organs which are morphologically infiltrated by leukaemic cells are significantly lower than in normal rats, while the persistence of measurable ADR concentrations does not change. On the contrary, ADR concentrations in organs not infiltrated by leukaemic cells are the same or slightly higher than in normal rats. Possible causes for these differences are either the differential properties of normal and leukaemic cells in their uptake and excretion of ADR, or anatomical and vascular changes. It is evident that, significantly different from normal. This observation may help in the prevention of toxicity by drug monitoring in serum.

Differences in the plasma kinetics of daunorubicin in normal and leukemic rats.

The plasma pharmacokinetics of the anthracycline drug daunorubicin in doses of 1 and 10 mg/kg body weight were compared in normal and leukemic inbred Sutton-August rats (50-70 x 10(9) leukemic cells/1 blood). The area under the plasma concentration versus time curve was about 25 times larger in the leukemic rats at both dose levels. The data indicate that daunorubicin has a lower volume of distribution in leukemic rats as compared with normals. The large reduction in plasma clearance of daunorubicin in the leukemic rats can to some extent be explained by impairment of hepatic and renal functions but the results indicate that the leukemic state per se was of importance for the observed pharmacokinetic differences. This finding may be of importance for the treatment of patients with a high leukemic cell load, since higher retention of the drug in the central compartment leads to lower concentrations in poorly vascularized tissues, which favours the development of resistant leukemic cell clones.

Examination of newly synthesized 2-chloroethyl-nitrosoureas on rat leukemia L 5222.

The preterminal intraperitoneal implanted rat leukemia L 5222 was used to test the chemotherapeutic activity of 10 newly synthesized nitrosoureas: ethylmethanesulfonato-CNU, acetamido-CNU, dihydroxypropyl-CNU, carboxyethyl-CNU, cyanoethyl-CNU, morpholino-CNU, piperidino-CNU, methylene-dioxybenzyl-CNU, methylene-3-pyridyl-CNU and methylene-4-pyridyl-CNU; their activity was compared against BCNU, CCNU, MeCCNU and hydroxyethyl-CNU. All compounds tested showed a more or less pronounced chemotherapeutic activity. MeCCNU was superior to all other compounds investigated. The predictive value for human tumors of results obtained from a model which is highly sensitive to nitrosoureas is discussed.

Monocytic origin of Fischer rat leukemia

Monocytic origin of Fischer rat leukemia

Effects of acronycine and of cytochalasin B on the division of rat leukemia cells

Acronycine (AN) is a lipophilic antineoplastic alkaloid which, like cytochalasin B (CB), inhibits nucleoside transport and causes binucleation through interference with cell cleavage. Unlike CB, AN causes swelling of membranous organelles and does not inhibit microfilament-mediated epithelial cell contractions. The effects of AN (6–12 μg/ml) and CB (1.5–4 μg/ml) on cell division were compared by direct observation and time-lapse cinemicrography in cultured IRC 741 rat leukemia cells. Both drugs caused dose-dependent binucleation and inhibition of the normal increase in cell numbers with the effects of 12 μg/ml AN approximating those of 4 μg/ml CB. In cells entering division after exposure to the drugs for up to 24 h, furrowing was completely inhibited in a dose-dependent proportion of cells by CB but not by AN. In cells where furrowing occurred, the intervals from the onset of anaphase to furrow initiation were shortened significantly by 1.5–3 μg/ml CB, but not by 4 μg/ml CB; they were also shortened after 4/2–6 h of 12 μg/ml AN, but were prolonged up to 60% by 7–24 h of 12 μg/ml AN. Intervals from furrow initiation to daughter cell separation were prolonged up to 50% by AN and 140% by CB. The intervals from onset of anaphase to nuclear membrane reconstitution were not affected by AN for up to 18 h and lengthened thereafter; they were shortened by CB after 3–19 h, depending on the dose. In AN-treated cells, single furrows formed and cleavage was accompanied by twisting and exaggerated elongation along the long axis of the dividing cells, while CB-treated cleaving cells were characterized by surface blebbing and the formation of several consecutive incomplete furrows. Most CB effects occurred rapidly, while AN effects were delayed. The results suggest that the modes of action of CB and AN differ and support other data indicating that AN interferes primarily with the structure, function and/or turnover of cell membrane components.

Different modes of mesenteric infiltration displayed by two rat leukemias. A study by scanning and transmission electron microscopy and by microcinematography.

Infiltration of the mesentery after intraperitoneal implantation of two transplantable rat leukemias, the undifferentiated L5222 and the myeloid BNML, was studied by means of scanning and transmission electron microscopy, and microcinematography. In animals implanted with L5222 cells, contraction of the mesenteric mesothelium is a conspicuous feature. It occurs within the first 24h after implantation and influences decisively the course of infiltration. In contrast, The presence of BNML cells leads to mesothelial contraction only in the terminal stage and, therefore, exerts no direct effect on infiltration. In addition, the two leukemias differ with regard to their cellular motility. Whereas L5222 cells locomote within the mesentery, only stationary movements are recorded with BNML cells. Based on the different interactions with the mesothelium and cell motilities, two distinct modes of infiltrating the mesentery could be ascertained for the two rat leukemias.

Evidence for the cellular origin of Gross virus-induced leukemia in the rat: description of a unique LDH isozyme sub-band in leukemic lymphoid cells and lymphohemopoietic precursor cells.

Neoplastic thymocytes from rat thymic lymphoma-leukemias induced by the rat-adapted Gross-leukemia virus (RAGV) were analyzed for a variety of differentiation markers to define their differentiation state and possible cellular origin. A majority of thymocytes from leukemic rats had the phenotypic characteristics of subcapsular cortical thymocytes that are the most ancestral of the thymocytes. These cells exhibited readily detectable levels of Thy-1 and histocompatibility antigens on their surfaces, they contained terminal deoxynucleotidyl transferase (TdT) and they contained low adenosine deaminase (ADA) and high purine nucleoside phosphorylase (PNP) specific activity. The leukemic thymocytes also contained a sub-band of the LDH-5 isozyme (LDH-5') that was not detected in normal thymocytes but that was present in lymphocyte-rich fractions of postnatal bone marrow, fetal and prepubertal spleen, and fetal and neonatal liver. The tissue distribution and ontogeny of LDH-5'-containing cells is similar to prethymic TdT+ cells in the rat and both TdT and LDH-5' are enriched in a subset of bone marrow "null" cells. These results suggest that TdT+ thymocyte progenitors or their precursors are the targets of leukemic transformation of RAGV.

Mechanism by which antibodies to non-AgB antigens mediate rejection of rat leukaemia cells.

The August and Hooded rat strains are compatible at the major histocompatibility locus (both are AgB5 or Rtlc). Antisera against the minor histocompatibility antigens of Hooded rats were raised by immunizing August rats with grafts of tumours or normal tissue. Such antisera, if transferred to normal unimmunized August rats, cause them to reject i.v. administered Hooded rat leukemia (HRL) cells within a few hours, and X-irradiated August rats, for whom a graft of HRL is lethal, can survive indefinitely if pretreated with the antiserum. The distribution of 125I-labelled HRL cells in the tissues of August rats was followed at times after their injection, and it was found that, in the presence of antiserum, i.v. administered leukaemic cells are rapidly destroyed in the liver and spleen. The active component of the antiserum is IgG antibody, and its action is independent of the lytic elements of complement. Antibody-mediated splenic and hepatic clearance of the leukaemia cells is unaffected by total-body X-irradiation but reduced by treating the rats with colloidal carbon. The data are consistent with the hypothesis that the rejection of HRL across the histocompatibility barrier studied is, in the presence of antibody, effected by immunophagocytosis.

A Rationale for Sequential High-Dose Chemotherapy of Leukemia Timed to Coincide With Induced Tumor Proliferation

Studies in leukemic rats (LBN-ML) tested the hypothesis that an enhanced effect of drugs in sequence relates to temporally predictable induced tumor growth. As leukemia progresses, tumor labeling index (LI) declines in association with increasing tumor-associated inhibitory activity (TAIA) in serum. Graded initial doses of arabinofuranosyl cytosine (ara-C) (50-350 mg/kg every 8 hr × 6) produced relative increments of induced humoral stimulatory activity (HSA) and LI in vivo by day 6, while overcoming the antiproliferative effects of TAIA. Rats bearing late-stage tumor with high TAIA and low LI were treated with 50 or 100 mg/kg ara-C every 8 hr × 6 on days 0,1 and on other days in sequence (0,1-0,1 through 0,1-14,15). Serum, WBC and tumor LI were measured. With the 50 mg/kg initial dose, TAIA was not overcome, and the LI did not achieve levels above pretreatment. A second course of ara-C in sequence produced only additive survival, regardless of timing. In contrast, with the 100 mg/kg sequence, TAIA was overcome, the LI increased, and synergistic survival occurred in those treated with the second ara-C on days 6,7 (760% of controls) at the time of peak HSA and tumor LI. Survival times in other groups in sequence were only additive. Incremental doses of ara-C on day 6 produced linear prolongation of survival. These studies demonstrate that TAIA correlates with tumor LI and that the degree of resultant tumor growth relates to the initial drug dose, tumor kill, and TAIA reduction, and the amount of HSA induced. Survival relates directly to the dose of the second drug given at this predictable peak of maximal HSA and LI.

Clearance of immune complexes formed in normal and leukaemic rats.

Immune complexes of 125I-HSA-rat anti-HSA formed in vivo under conditions of antibody excess were rapidly cleared from the circulation of both normal and leukaemic Hooded rats. In HSA-immune rats most of the 125I-HSA present in the blood was found to be cell-bound, but a proportion was present as circulating immune complexes that could be precipitated from plasma by 2.5% polyethylene glycol. There was no evidence that clearance of a soluble antigen was impaired in leukaemic animals.

Chemotherapy of autochthonous leukemias induced by 7,12-dimethylbenz(a)anthracene in Long Evans rats.

A total of 111 autochthonous (primary) acute rat leukemias of the diffuse-hepatic type which had been induced by 7.12-dimethylbenz(a)anthracene were treated with mono- and combination chemotherapy. The survival time of treated rats increased significantly compared to untreated controls. A combination of vincristine (VCR), adriamycin (ADR), and cytosine arabinoside (Ara-C) was superior to a combination of VCR and ADR as well as to a monotherapy with Cyclophosphamide, BCNU, VCR, ADR, and Ara-C.

Inhibition of spontaneous transformation of rat embryo cells releasing endogenous type C virus by virus-specific antiserum.

Noninbred Sprague-Dawley rat embryo cell clones predictably undergo transformation after 20-30 in vitro passages following spontaneous release of endogenous rat leukemia virus (RaLV). In the presence of RaLV-specific antiserum, virus production and infectivity were reduced and transformation was delayed from 6 to 25 weeks. Transformation was not associated with an increased expression of Kirsten murine sarcoma virus-related src gene RNA.

Examination of four newly synthesized 2-chloroethylnitrosoureas in comparison with BCNU, CCNU, MeCCNU, chlorozotocin and hydroxyethyl-CNU in preterminal rat leukemia L 5222

The newly synthesized nitrosoureas 1-(2-chloroethyl)-1-nitroso-3-(methylenecarboxamido)-urea (Acetamido-CNU), 1-(2-chloroethyl)-1-nitroso-3-(4-morpholino)-urea (Morpholino-CNU), 1-(2-chloroethyl)-1-nitroso-3-(1-piperidino)-urea (Piperidino-CNU), and 2-[3-(2-chloroethyl)-3-nitrosoureido]-ethylmethanesulfonate (Ethylmethanesulfonato-CNU) were compared in their chemotherapeutic activity against preterminal rat leukemia L 5222 with BCNU, CCNU, MeCCNU, Chlorozotocin, and Hydroxyethyl-CNU. With respect to the dose range effecting a median survival time of more than 90 days, MeCCNU was superior to the other substances. Chlorozotocin, on the other hand, was the only substance which achieved no cures in this experimental arrangement. From the four newly introduced substances the water-soluble substances Acetamido-CNU and Morpholino-CNU were approximately comparable to CCNU with regard to the dose range effecting a median survival time of greater than 90 days. Piperidino-CNU and Ethylmethanesulfonato-CNU also effected cures; however, only Piperidino-CNU in one dosage effected a median survival time of greater than 90 days.