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.
Six laboratories performing estrogen receptor analysis in breast cancer for patients included in clinical protocols of the Eastern Cooperative Oncology Group participated in a quality control study for estrogen receptor assay. Three tissue powders containing 10–50 femtomoles of estrogen receptors/mg cytosol protein were distributed and the results analyzed for interlaboratory and intralaboratory variations. Frequency of misclassification of the receptor-positive powders as receptor-negative was related to the quantity of receptors. All labs except one were able to distinguish “low content” from “medium content” and the “medium” from the “high content” powders despite the variability in the numerical values reported for the powders. Two of the six labs reported consistently low values for all samples and the results of one lab were consistently higher than the average of the values obtained for the powders. These results could not be explained by the variations in protein content reported by these laboratories for different powders. Implications of the degree of inter-lab and intra-lab variations in the assay results are discussed with reference to their effect on selecting patients for hormonal vs chemotherapy. Based on the results of this study, tentative suggestions for quality control for the receptor assay are also made.
Hormone-responsive mammary tumors of GR mice were treated with tamoxifen, cyclophosphamide, or with both drugs combined. Tamoxifen alone or cyclophosphamide alone caused inhibition of tumor growth, but more growth inhibition was obtained with the combined therapy.
Several dimethylbenz[c]acridines and their isomers dimethylbenz[a]acridines were studied for their capacity to induce malignant transformation in secondary hamster embryo cells. Transformation was evaluated by the ability of transformed cells to provoke tumor formation in syngenic animals. 7,8-Dimethylbenz[c]acridine, 7,10-dimethylbenz[c]acridine, as well as a mixture of both, caused malignant transformation of hamster embryo cells, but not 7,9-dimethylbenz[c]acridine. Cultures treated with 10,12-dimethylbenz[a]acridine and 9,12-dimethylbenz[a]acridine did not become malignant and showed a decreased life-span in vitro. Untreated control cells retained their original characteristics and when injected in animals did not produce tumors throughout the whole experiment.
A phase II evaluation of chlorozoticin (CZT), a water soluble nitrosourea, was undertaken to determine its effectiveness and toxicity in a variety of human metastatic neoplasms. The dosage regimen chosen was either 90 or 120 mg/m 2 given by i.v. bolus every six weeks. Dosage escalation or de-escalation was dependent on toxicity. There have been 152 patients evaluable for response. The only significant response rates observed were in non-Hodgkin's lymphoma ( 5/11 ) and sarcoma ( 4/27 ). Single responses were observed in breast and oat cell carcinoma of lung. No responses were observed in melanoma, colorectal, kidney, non-oat cell lung, pancreas, stomach and other carcinomas. Hematological toxicity has been minimal as predicted, but does appear to be cumulative. The major G.I. toxicity has been nausea and vomiting—usually controllable. Occasional hepatic enzyme elevations were observed, and azotemia was observed in 6 patients. Both were reversible. Rare skin and occasional CNS reactions were also seen.
2,3-Dihydro-1H-imidazo [1,2-b] pyrazole (IMPY, NSC-51143) is a new ribonucleotide reductase inhibitor, presently undergoing clinical evaluation, that exhibits prolonged in vivo antitumor activity in experimental animals. Disposition studies were initiated to determine if the prolonged in vivo antitumor activity of IMPY could be explained by its pharmacologic properties. Plasma disappearance curves of radioactivity were biphasic after the i.v. administration of 100, 250 or 500 mg/kg to rats and 250 mg/kg to mice. The distribution phase was rapid in each case (t12 of approximately 30 min or less), followed by a prolonged elimination phase. Radioactivity was distributed to all tissues of rats and mice including brain after an i.v. dose of 250 mg/kg. In rats, 67.8% of the administered radioactivity was excreted in the urine during the first 24 hr. By 120 hr 74.3% had been excreted via the urine compared with 11.1% in the fees, the latter by biliary excretion. The chromatographic profile of the urine collected from rats and mice 4 hr after drug administration indicated extensive metabolism. Thus, the prolonged plasma and tissue levels of parent IMPY and its metabolites can account for the prolonged duration of cytotoxic activity in vivo.
The total number of lymphocytes and the number and proportion of high-(29°C) and low- (4°C) affinity E (erythrocyte) rosette-forming cells (RFC) from the peripheral blood of 33 patients with Hodgkin's and 41 patients with non-Hodgkin's lymphoma was assessed, and compared with results obtained from normal controls. Analysis of results was performed on groups of patients subdivided according to age, histological type and stage of disease. Patients with Hodgkin's disease showed a decrease in RFC which was attributed to a reduction in low-affinity RFC; this decrease could not be related to any particular histological type or stage of disease. Patients with non-Hodgkin's lymphoma showed, in addition to a decrease in low-affinity RFC, a significant decrease in high-affinity RFC. The observed decrease in peripheral blood T cell numbers of non-Hodgkin's lymphoma patients did not correlate with the type of disease, but patients with clinical stage IV showed a more pronounced reduction in the number of high- and low-affinity RFC when compared with other groups.
N-Formyl-leurosine labeled with 14C in the formyl group was administered to six patients with malignant disease, and the pharmacokinetic behavior of the drug was determined. Chromatographic studies on plasma showed the presence of unchanged drug and 7 metabolites. No metabolites were found in the urine. The plasma decay curve of the unchanged drug was biphasic with half-life values of t12α = 18.6 min and t12β = 4.28 hr. Within 72 hr only 30–40% of the radioactivity could be recovered in urine and feces in 5 patients. One patient consumed laxatives during the treatment. In this case 95% of the 14C-dose was recovered. The contribution made by fecal elimination was 80%.
A pulse of the protein synthesis inhibitor cycloheximide is shown to induce chromosome aberrations in cultured cells of human origin. These aberrations do not appear until in excess of 12 hr after the pulse and their appearance coincides with the time when cells which had been in S-phase at the time of the cycloheximide pulse reach mitosis. We suggest that the reason why the protein synthesis inhibitor cycloheximide has previously been reported not to induce chromosome aberrations is due to the long delay of cells with damaged chromosomes in reaching mitosis.
Coadministration of Tween 80 enhances the activity of adriamycin against selected experimental tumors in mice. Although some investigators have suggested a direct effect of Tween 80 on tumor cells, we wished to determine whether altered plasma concentrations of adriamycin occurred which might account for the apparent therapeutic synergism. Male CDF1 mice were treated i.p. with 6.7 mg/kg of adriamycin alone or combined with 5000 mg/kg of Tween 80 in physiologic saline. Fluorometric determination of adriamycin equivalents in plasma revealed significantly higher adriamycin concentrations 1 and 2 hr post treatment in mice that had received Tween 80. In three separate experiments, a significant, reversible increase in packed cell volume occurred in the peripheral blood of mice treated i.p. with Tween 80. This increase was maximal at 1–2 hr post treatment. The magnitude of the apparent plasma volume reduction accounted quantitatively for the increase in drug concentration.
We describe the synthesis and some biological properties of N-(4-N-[2-amino-4-hydroxy-6-quinazolinyl)methyl]prop-2-ynylaminobenzoyl)-l-glutamic acid (CB 3717), a potent antifolate inhibitor of thymidylate synthetase (EC 2.1.1.45). This compound inhibited thymidylate synthetase from L1210 cells competitively with respect to the substrate methylene-tetrahydrofolate. The inhibitor constant (Ki) was approximately lnM. Toxicity to L1210 cells in suspension culture could be reversed more effectively by co-incubation with thymidine than with folinic acid. A cultured subline of L1210 cells resistant to methotrexate by virtue of increased cellular dihydrofolate reductase (EC 1.5.1.4) content was not cross-resistant to CB 3717. Treatment of animals bearing the L1210 tumour with CB 3717 at 125 or 200 mg/kg/day for 5 days resulted in ‘cures’ (>120 day survival) in 90% of animals. These doses did not cause obvious toxicity or significant weight loss in animals. The studies in tissue culture and the enzymology suggest that thymidylate synthetase is the cytotoxic locus of action of this compound. The high therapeutic efficacy of CB 3717 may be due to preservation of de novo purine synthesis, which is inhibited by other antifolates. The in vitro results further suggest that CB 3717 could be active against methotrexate-resistant tumours.
Significant enhancement of tritiated dCyd suicide occurred when unlabelled dThd was added to cultures of mouse monocytic colony-forming cells. Incorporation experiments supported the suicide experiments in that incorporation of tritiated dCyd into DNA was significantly increased. One hundred micromolar dCyd significantly reduced the radiotoxicity of 0.3 μCi of tritiated dThd; incorporation experiments indicated a dose-related reduction in the incorporation of tritiated dThd into DNA with the addition of 1–100 μM unlabelled dCyd. The addition of 1 μM aminopterin reversed the effect of 100 μM deoxycytidine; viz., incorporation of dThd into DNA was 90% of controls. Aminopterin had a similar effect on deoxyuridine reversal of tritiated dThd incorporation into DNA. Aminopterin had no effect on the reduction of tritiated dThd incorporation into DNA due to the addition of 100 μM unlabelled thymidine. Unlabelled ribonucleosides, Urd and Cyd, did not significantly affect the suicide pattern of tritiated dThd or dCyd when they were added to CFC cultures. Unlabelled deoxyribonucleosides, dThd or dCyd, did not significantly affect the suicide pattern of either tritiated Cyd or Urd when they were added to cultures containing tritiated ribonucleosides. Unlabelled Urd or Cyd was effective in reversing the suicide due to tritiated Urd or Cyd.
Two hundred and forty-two patients received 1200 doses of cis-diamminedichloroplatinum (II), generally out of the hospital, at 50 mg/m2 every 3–4 weeks. Furosemide and mannitol were given to assure a brisk diuresis when drug was administered, and 2 litres of 0.45% saline-5% dextrose were given over two hours to assure hydration. Azotemia developed after 15 courses (1.3%) in eleven patients (4.5%). Peak serum creatinine was 2 mg/dl in only four patients, and azotemia lasted 12 weeks in only one episode. Incidence of azotemia did not increase with increasing cumulative dose. Two patients had allergic reactions, one died suddenly during drug administration, three had clinically evident hearing loss, and nearly all patients had moderately severe vomiting. Peripheral neuropathy occurred in only 1 of 155 patients not given concomitant hexamethylmelamine.
The accumulation in rat heart ventricles, the presence of metabolites and the subcellular localization of daunorubicin (DNR), doxorubicin (DOX) and their DNA complexes were investigated. After intravenous perfusion of free DNR or DOX, equal amounts of the drugs are recovered in the heart, whereas after injection of their DNA complexes, the accumulation levels represent 42% (DNR) and 32% (DOX) of those reached with the free drugs. Daunorubicinol and daunomycinone are detected after administration of DNR or DNR-DNA but only trace amounts of metabolites are found with DOX and DOX-DNA. Subcellular distributions of DNR injected as free or DNA complex are similar. Isopycnic and differential centrifugations indicate that DNR could be associated with nuclear DNA and to a smaller extent with lysosomes. By contrast, after injection of free or complexed DOX the drug is almost exclusively associated with the nuclear DNA. The mechanisms by which these drugs are accumulated by the heart and the relation with their cardiotoxicities are discussed.
These studies were conducted to determine whether the metastatic heterogeneity that is frequently observed in primary neoplasms is a consequence of multicellular transformation or acquired genetic variability. BALBc embryo fibroblasts were infected in vitro with mouse sarcoma virus. Six tumor colonies, each derived from a single transformed cell, were isolated and propagated as individual cell lines. Twenty-four days after virus infection, mice were injected s.c. or i.v. with viable cells harvested from the individual lines. Subcutaneous tumors developed in nearly all of the mice and regressed 30 days after inoculation. In contrast, the production of lung tumor colonies varied significantly among the cell lines. Moreover, the individual lines were found to be heterogeneous. This conclusion is based on results of experiments in which two cell lines exhibiting either a low or high propensity to produce lung tumor colonies were subcloned. Cells from these subclones were injected i.v. into syngeneic mice. The subclones differed significantly among themselves and from the parent culture in their ability to produce lung tumor colonies. We conclude that regardless of whether neoplasms are unicellular or multicellular in origin, they can be heterogeneous and contain subpopulations of cells with different metastatic properties by the time of diagnosis.
Cell-specific G1-inhibitory (chalone) activity has been extracted from old JB-1 ascites tumors and purified by means of G-15 Sephadex chromatography. Four peaks of activity were obtained, the amount of activity in each peak varying from batch to batch. The first eluting activity peak probably represents chalone activity present in an aggregated form. The latest eluting activity peak was totally unspecific and could be attributed to the presence of high amounts of salts. The two intermediary peaks of activity were investigated in more detail. It is shown that the first eluting activity peak of these two is due to the polyamine spermine complexed to a carrier. Although the spermine complex exhibits a certain degree of cell-specific inhibitory activity in vitro, it is totally inactive in vivo. The second eluting activity peak containing the main part of cell-specific G-inhibitory activity has been characterized as a small molecular weight (Mr300–600), ampholytic, hydrophobic, slightly acidic, and thiol-containing peptide active both in vitro and in vivo.
Male C3H mice bearing transplanted tumours were treated with hyperthermia, γ-irradiation and the radiosensitising drug misonidazole. The activity of tumour lysosomal acid phosphatase and β-glucuronidase was determined using quantitative cytochemical techniques which measure both lysosomal membrane permeability and enzyme activity. Misonidazole (0.5 mg/g) had no effect on the membrane permeability or enzyme activity of tumour lysosomes 1 hr after injection; but 25 hr after the drug treatment the permeability of the lysosomal membrane to the substrate was increased to 1.7 times control. Increases in the lysosomal enzyme activity and membrane permeability were, however, observed 1 hr after combined treatment with misonidazole and irradiation, although neither the drug nor irradiation given alone affected the lysosomes 1 hr after treatment. Twenty-five hours after treatment of tumours with 0.5 mg/g misonidazole given 25 min before irradiation of tumours with 1 krad, permeability of the lysosomal membrane had increased to 2.3 times the control. The effects of the irradiation and the radiosensitisers were thus synergistic. Hyperthermic treatment of tumours at 43°C for 1 hr caused increases in the lysosomal membrane permeability and enzyme activity measured immediately after exposure, but misonidazole reduced both membrane permeability and enzyme activity. These experiments have demonstrated that misonidazole and irradiation act synergistically to cause increased lysosomal activity, but that misonidazole depresses the effect of hyperthermia on lysosomes.
Measurements of serum concentrations of DNA and the DNA-binding protein C3DP by radioimmunoassay showed that the levels of both substances tend to increase in cancer patients during active malignant disease. In most cases, the levels returned to normal during chemotherapy-induced remission; however, the changes in concentration for DNA and C3DP did not occur simultaneously, and no correlation was found between their levels. The sera of cancer patients contained a strong inhibitor of DNAse. We examined the possibility that C3DP may have such an inhibitory effect by binding to DNA and preventing the action of DNAse. The enzyme was fully active in the presence of purified C3DP, indicating that the DNAse inhibitor in cancer serum was a substance other than C3DP. Although the relationship between DNA and the DNA-binding protein remains unknown, their measurement may have diagnostic and prognostic value.
A transplanted murine ovarian tumor (M5076/73A, M5, of C57B16 origin) with unique metastatic potential is described. After i.m. or s.c. inoculation of the M5 ovarian tumor, spontaneous metastases were consistently observed in liver, spleen, kidney, ovary and uterus, but lung lesions were not usually found. Even after i.v. inoculation the tumor showed a selective tropism for the abdominal organs mentioned. When M5 ovarian carcinoma cells were injected i.p., the tumor grew as scattered lesions in the abdomen and resulted in carcinomatous ascites. Surgical excision of i.m. tumors as early as 5days after implantation failed to protect mice against metastases, suggesting early widespread dissemination from the primary lesion. The tumor was poorly immunogenic as assessed by in vivo immunization with irradiated cells and by concomitant immunity experiments.
The radiation response of two human malignant melanomas (V.N. and G.E.) irradiated as solid tumours in athymic nude mice was studied by measuring single cell survival in soft agar. Survival curves were determined for cells from tumours irradiated either in air-breathing mice or in mice killed by cervical dislocation 15 min before irradiation. The hypoxic fractions, determined from the vertical displacement of these two curves, were 12–17% for V.N. melanoma and 26–36% for G.E. melanoma. The D0-values were 3.43 Gy (air-breathing mice) and 3.05 Gy (dead mice) for V.N. melanoma and 3.00 Gy (air-breathing mice) and 3.35 Gy (dead mice) for G.E. melanoma. Survival curves of cells irradiated in dead mice showed n = 4.6 and Dq = 4.6 Gy for V.N. melanoma and n = 2.0 and Dq = 2.2 Gy for G.E. melanoma.
