ALTERATIONS IN 3H-THYMIDINE INCORPORATION INTO DNA INDUCED BY METHYL CCNU [1-(2-CHLOROETHYL)-3-(4-METHYL CYCLOHEXYL)-1-NITROSOUREA] IN NORMAL AND TUMOROUS TISSUES IN VIVO

Methyl CCNU produces a suppression of tritiated thymidine (³H-TdR) incorporation into DNA in vivo in normal bone marrow and gastrointestinal tissues which is different in magnitude and duration from that seen in L1210 ascites tumor in the same animals. This suppression and recovery pattern is not seen in animals bearing L1210 ascites tumor resistant to MeCCNU. Where a different pattern of recovery is seen between normal host target tissues and tumor, the pattern can be exploited to increase the cure rate of animals bearing advanced L1210 ascites tumor with properly spaced second doses of MeCCNU. Additional information on the potential toxicity of second doses of MeCCNU can be predicted from knowledge of the time of recovery of DNA synthesis in the normal host target tissues.     

THE EFFECT OF METHYL CCNU (NSC-95441) ON THE CELLULAR KINETICS OF NORMAL AND LEUKEMIC MURINE TISSUES IN VIVO

The effect of MeCCNU on the cellular kinetics of normal and leukemic murine tissues is examined in vivo . A prolongation in the S phase is noted after doses of MeCCNU as low as 4 mg/kg, and this prolongation persists for approximately 4 hr after the dose. No persistent alteration in the growth fraction is produced by MeCCNU. The generation time of the cells regrowing after MeCCNU is slightly prolonged, compared to normal L1210 leukemia. Simultaneous studies on ascites tumor, normal bone marrow, and normal gastrointestinal mucosa, using the in vivo uptake of ³H-thymidine into DNA, have shown a differential effect on the tumor and normal tissues.     

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU)/interleukin-2 chemoimmunotherapy of murine L1210 leukemia

Summary We have developed a chemoimmunotherapy regimen for the treatment of L1210-cell-induced ascites tumors in mice using a combination of sub-toxic doses of interleukin-2 (IL-2) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU is administered intraperitoneally 4 days after tumor implantation and followed 2 days later by single doses of human recombinant IL-2 for 3 consecutive days. An optimum survival of 84% was achieved using 1500 U IL-2. Reduced survival was observed when lower or higher IL-2 dosages were used. No therapy resulted when heat-inactivated IL-2 was used or when IL-2 was used without chemotherapy. Surviving animals were resistant to L1210 leukemia but not P815 mastocytoma tumor challenge suggesting the combined BCNU/IL-2 therapy stimulated tumor-specific immunity.     

Disruption of DNA synthesis and structure of mouse L1210 leukemia cells, sensitive and resistant to 1-methyl-1 nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea in vivo

Leukemia L1210 cells with acquired resistance to 1-methyl-1-nitrosourea (MNU) (L1210/MNU) and 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (L1210/BCNU) were developed from leukemia L1210 cells sensitive to these drugs (L1210/0). The modal chromosome number of leukemia L1210/MNU and L1210/BCNU cells increases from 40 (L1210/0) to 41. It was shown that in leukemia L1210/MNU cells the inhibition of DNA synthesis after MNU administration in a therapeutic dose (80 mg/kg) is lasted within 24 hours, while that in leukemia L1210/0 cell--within 96 hours. After administration of BCNU (20 mg/kg) inhibition of DNA synthesis in leukemia L1210/BCNU cells reached of 50% of control in comparison with practically complete inhibition of DNA synthesis in leukemia L1210/0 cells. Centrifugation on alkaline sucrose density gradients revealed no differences in the rate of sedimentation of leukemia L1210/0, L1210/MNU and L1210/BCNU cell lysates. After 1 hour treatment with MNU of mice bearing L1210/MNU and L1210/0 leukemia cells single-strand breaks in DNA were determined. After 4 hours these strand-breaks retained in leukemia L1210/0 cells, but were eliminated in leukemia L1210/MNU cells. Administration of BCNU to mice with leukemia L1210/0 and L1210/BCNU cells resulted in both cases in the production of DNA aggregates. There is no complete cross-resistance between MNU and BCNU which allows a substitution of these drugs providing for the increase in their therapeutic efficiency.     

Polyamines antagonize both the antileukemic activity and the reverse transcriptase stimulatory activity of 4,4'-diacetyldiphenylurea bis(guanylhydrazone) (DDUG)

(Diacetyldiphenylurea)bis(guanylhydrazone) (DDUG) functions as a cationic trypanocide antagonized in vivo by exogenous concomitant addition of the biologically active polyamine, spermine. It also inhibits the DNA polymerases of L1210 murine leukemia cells. We have found that DDUG stimulates Rauscher murine leukemia virus DNA polymerase activity in a manner similar to polyamines. Such stimulation does not occur if DNA synthesis is carried out on spermine + activated DNA complexes. We also show that the in vivo antileukemic activity of DDUG in the L1210 ascites mouse model is antagonized by biologically active polyamines. These studies suggest a new intracellular target for the antileukemic activity of DDUG: interference with polyamine function.     

Comparative effects of Yoshi-864 and busulfan on certain transplantable murine tumors.

Yoshi-864 extends markedly the survival times of mice bearing L1210 leukemia or Ehrlich ascites carcinoma. Busulfan, with methanesulfonate leaving groups identical with those of Yoshi-864, is without effect. Tumor cells from mice bearing the Ehrlich tumor and treated with Yoshi-864 have a persistent reduction in ability to synthesize DNA. Synthesis of DNA in cells from mice treated with busulfan is moderately suppresed at 48 hr after treatment, but returns virtually to the control value at 72 hr.     

Relation between the changes in the structure and synthesis of DNA in the cells of mouse leukemia L1210 induced by 1-methyl-1-nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea

The damage of DNA structure and synthesis in murine leukemia L1210 cells upon single administration in therapeutic doses of antitumour agents of N-nitrosourea type, such as 1-methyl-1-nitrosourea (MNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was studied. MNU and BCNU were characterized by stronger inhibitory effects on de novo DNA synthesis compared to additional pathway of DNA synthesis in leukemia L1210 cells in vivo. Centrifugation in alkaline sucrose density gradients of L1210 cell lysates has revealed persistent single-strand breaks and alkaline-labile sites in newly replicated DNA. Parental DNA structure was more stable to damaging drug effects than that of newly replicated DNA. The results are consistent with our previous data on the differences in the mechanisms of MNU and BCNU action and the absence of complete cross resistance between the drugs.     

Effect of schisanhenol on the antitumor activity of adriamycin

Schisanhenol (Sal) did not diminish the antitumor activity of adriamycin in mice bearing P388 ascites tumor. Sal did not antagonize the suppressive effect of adriamycin on DNA synthesis and cell proliferation in an L1210 ascitic tumor cell culture. Furthermore, Sal at the concentration of 0.1, 0.25, or 1 mM accelerated adriamycin-dependent DNA damage in the presence of Fe3+ in vitro. It appears that Sal was able to protect against adriamycin induced heart mitochondrial toxicity, while it did not antagonize the antitumor activity of adriamycin.     

Morphological characterization of lymphoid cells conjugates with scattered tumor target cells in organ imprints of Yoshida ascites bearing rats

The authors analysed morphologically the ability of host effector cells to bind disseminated tumor target cells (pre-lysis) in vivo. Scattered tumor cells are morphologically visible stained with May Grünwald-Giemsa in the lung, liver, kidney, and spleen imprints of Yoshida ascites tumor bearing rats. Lymphoid cells can be seen binding disseminated tumor target cells in organs but not in the peritoneal cavity. This model may provide a useful technique for morphological studies on the in vivo conjugation capacity of host effector cells against tumor target cells.     

Enhanced Antibody-Dependent Lymphocyte-Mediated Cytotoxic activity in mice cured from an ascitic tumor by a single tumor aspiration

Summary Aspiration of approximately 50% of the tumor volume in the syngenic lethal C3H-L1 ascites tumor system at day 12 after intraperitoneal injection of 2×10⁷ tumor cells cured about 40% of the animals from the ascites tumor. The recovering mice showed strong immunity to a second challenge with tumor cells and transfer of spleen cells from recovering mice protected normal mice to tumor development. Complement-dependent serum-mediated cytotoxicity (CDSC) against tumor cells in vitro appeared both in non-recovering and recovering mice being most pronounced in completely cured animals. Antibody-dependent, lymphocyte-mediated cytotoxicity (ADLC) was absent in tumor-bearing and dying mice but appeared early after recovery in cured mice. No direct lymphocyte-mediated cytotoxicity (DLC) could be demonstrated either in non-recovering or in recovering mice.     

Effects of the bioreductive alkylating agent 2,3-bis(chloromethyl)-1,4-naphthoquinone on coupled mitochondria isolated from sarcoma 180 ascites cells.

The effect of CMNQ was studied on mitochondria isolated from S-180 ascites tumor cells. It was found that the primary metabolic event upon addition of CMNQ to S-180 mitochondria was a stimulation of oxygen uptake. The oxygen utilization rate was maximized at about 50 nmoles CMNQ/mg protein; at doses higher than this, inhibition of respiration was observed relative to the stimulation of respiration produced by CCCP. It was also up to 50 nmoles CMNQ/mg protein. S-180 ATPase activity is stimulated maximally by 125 nmoles CMNQ/mg protein; at doses higher than this, slight inhibition of the ATPase activity relative to the stimulation produced by CCCP is seen. In vivo treatment of CMNQ to tumor bearing animals leads to a significant reduction of in vitro S-180 cellular respiration rates. The data presented in this work coupled with previously published reports involving CMNQ support the proposal for a mitochondrial level of action for this bioreductive alkylating antineoplastic agent.     

Increases in mRNA levels of glucose transporters types 1 and 3 in Ehrlich ascites tumor cells during tumor development

A common feature of many tumors is an increase in glucose catabolism during tumor growth. We studied the mechanism of this phenomenon by using Ehrlich ascites tumor bearing mice as the animal model. We found that Ehrlich ascites tumor cells possess only glucose transporter 1 (GLUT1) and GLUT3 but no GLUT2, GLUT4, or GLUT5. The mRNA levels of GLUT1 and GLUT3 increased progressively in the tumour during development; however, there were no changes observable in mRNA levels of glucose transporters of all types in brain, liver, and heart of the host mice. These findings suggest that Ehrlich ascites tumor augments its glucose transport mechanism relative to other tissues in response to its unique growth needs. J. Cell. Biochem. 67:131–135, 1997. © 1997 Wiley-Liss, Inc.     

Dose-dependent cytokinetic changes following 1-beta-D-arabinofuranosylcytosine and hydroxyurea in L1210 and S-180 in vivo.

DNA synthesis inhibition and recovery in L1210 and S-180 ascites tumors following 1-beta-D-arabinofuranosylcytosine (Ara-C) and hydroxyurea (HU) were measured autoradiographically as a basis for optimizing drug schedules. Tumor bearing mice, 10(6) cells day 0, were treated on day 4 with 20, 200 or 2000 mg/kg Ara-C or 50, 300 or 1800 mg/kg HU. At various intervals following drug, [3H]thymidine was administered i.p. and mice were killed 1 hr later. Tumor cells were analyzed for labeling index (LI) and grain count (GC) to determine the percentage of cells in S phase and the distribution of DNA synthesis rates among the labeled cells, respectively. Following each dose of HU, DNA synthesis was inhibited completely. Recovery of LI was rapid and approached control values by 6 hr. Following each dose of Ara-C, DNA synthesis was inhibited completely for at least 6 hr. Recovery of LI was first noted 6 hr following 20 mg/kg Ara-C and 9 hr following 200 mg/kg. Following both doses the LI reached 100% of the control value by 26 hr. GC analysis indicated that following Ara-C treatment, DNA synthesis was reinitiated first with cells with low GC from 6 to 12 hr followed by cells with increasing GC from 12 to 20 hr. The labeling intensity reached control values by 20 hr and an 'overshoot' occurred by 26 hr. These data suggest that the recovery of DNA synthesis rate is a gradual process. Survival data for mice receiving two doses of Ara-C indicated that the optimal interval for retreatment following the lower dose of Ara-C occurred by 6 hr as compared to 12--16 hr for the higher dose. These times coincided in both instances with recovery of LI to 33--50% of control values. Early recovery of LI may be the best method currently available for estimating the optimal time for retreatment with an S phase specific drug.     

Comparison of nuclear DNA-dependent rna polymerases from mouse tumors and tissues of normal swiss mice

• 1.1. Five major forms of nuclear DNA-dependent RNA polymerases from seven transplantable murine tumors and from five tissues from normal, adult, male Swiss mice (Mus musculus) were separated chromatographically on DEAE-Sephadex A-25. Forms Ia and Ib were insensitive to α-amanitin, whereas forms IIa and IIb were highly sensitive and form III was slightly sensitive.
• 2.2. The polymerases from all tumors or ascites tumors only were compared statistically with those from the normal tissues in regard to elution patterns, specific activities, activities per mg of nuclear DNA. degrees of purification and yields. Similarly, individual tumors were compared with homologous normal tissues. All parameters were characterized by relatively large variance.
• 3.3. Activities of all RNA polymerases per mg DNA were higher in all tumors compared with normal tissues. The order of statistical significance of these differences was lib > III > IIa > Ib > Ia.
• 4.4. Activities per mg DNA of all RNA polymerases from 6C3HED and L1210 tumors, with the exception of L1210 tumor enzyme III, were significantly greater than those from spleen, but only the activities of Lewis lung tumor enzyme IIb and of Taper hepatoma enzymes Ia and III were higher than those from the homologous tissues, lung and liver, respectively.

     

Development of host-dependent high-grade tumor-specific immunity through a novel mechanism triggered by the Lyt-2+ tumor-specific T cell clone (K7L) that induces temporal growth of L1210 leukemia-K7L-variant

When a murine leukemia L1210-specific Lyt-2+ T cell clone, K7L, was injected i.p. into CD2F1 mice together with L1210, the normal growth of L1210 in the peritoneal cavity of the mice at the early stage (days 0 to 5) was strongly inhibited, but L1210 grew progressively at the middle-stage (days 5 to 10), and then was rejected at the late stage (days 10 to 20). The mice thus survived for long times (more than 60 days), whereas the normal control injected with L1210 alone died within 14 days. The L1210 that grew at the middle stage in mice initially inoculated with L1210 together with K7L was a K7L-insensitive (K7L-) variant. All of eight tumor clones established from L1210-K7L- by limiting dilution was insensitive to the antitumor activity of K7L, and this property of tumor clones was stable after repeated in vitro passage. The initial depression of the L1210 growth by K7L followed by growth and rejection of the variant L1210-K7L- by the host T cell activity was then found to prepare a strong, long-lasting (more than 3 mo) immunity to protect mice against the high-dose (10(7) cells per mouse) challenge of original L1210. Corresponding to this result, definite tumor (L1210)-specific cytotoxic T lymphocyte (CTL) activity against both variant and original L1210 targets was developed by antigen (L1210) restimulation in the culture of spleen cells from these mice, but was not increased to a detectable level before L1210-K7L- variant started to grow. It was suggested that the 1210-K7L- variant and the original L1210 should have the common tumor-specific antigen that was independent of the K7L-reactive antigen, and that original L1210, whose growth was retarded by K7L, primed the host with the common antigen to be enormously boosted by the subsequently growing L1210-K7L- variant.     

CP-46,665-1: A novel lipoidal amine with antimetastatic and immunomodulatory properties

Summary CP-46-665-1 [4-aminomethyl-1(2,3-(di-n-decyloxy)-n-propyl)-4-phenylpiperidine dihydrochloride] is a novel, low-molecular-weight, synthetic compound with antimetastatic activity in rodent models. In the B-16 melanoma/C57Bl/6 mouse system, in which the hind limb bearing a primary foot tumor is amputated 21 days after tumor implantation, intravenous infusion of CP-46,665-1 after surgery at doses ranging from 0.15–2.5 mg/kg increases the fraction of metastasis-free animals at 50 days to as high as 56% (significantly different from the 19% seen in surgery-only controls, P<0.001). In more preliminary experiments with a mammary adenocarcinoma (13762)/Fischer 344 rat system, there are suggestions that the combination of CP-46,665-1 administration and surgical removal of the primary tumor results in increased numbers of long-term survivors that are free of metastatic disease.The mechanisms underlying the antimetastatic activity of CP-46,665-1 are not fully evident, but certain lines of evidence suggest that the compound may act by affecting the reticuloendothelial system (RES). Peritoneal macrophages from animals dosed with CP-46,665-1 display elevated cytolytic activity against tumor target cells. Further, radiolabeled CP-46,665-1 and/or its metabolites preferentially accumulate in organs of the RES, and lymph nodes, spleens, and livers from animals dosed chronically with CP-46,665-1 contain scattered foci of histiocytic cells with foamy vacuoles containing material staining as complex lipid. CP-46,665-1 augments the vaccine potency of irradiated L-1210 leukemia cells, but does not induce interferon in rodents.CP-46,665-1 appeared to be well tolerated in rats and dogs when infused at doses of up to 25 mg/kg at 4-day intervals for 92 days. In addition to the changes in the lymphoreticular system described above, the principal findings in dogs that received high doses by injection were erythrocyte lysis accompanied by the expected serum chemistry and hematological correlates; microcytic anemia and injection site irritation were found in rats receiving high doses. Neither species displayed notable toxic reactions when chronically dosed at the 1 mg/kg level, a level well within the antimetastatic activity dose range. Cats and dogs with a variety of malignancies tolerated weekly doses of the agent as high as 5 mg/kg with minimal side-effects for periods up to 30 weeks. We believe that this combination of good chronic toleration and potent antimetastatic activity commend CP-46,665-1 well for more extensive study as an experimental drug for the treatment of cancer.     

The role of N-nitrosourea-induced changes in the nucleoid structure and activity of repair enzymes in the development of drug resistance in mice with leukemia L1210

Using centrifugation of the nucleoid in a neutral sucrose gradient, the damages in the secondary structure of DNA and the activity of repair enzymes, such as DNA-polymerases alpha and beta and poly(ADP-riboso) polymerase, induced by 1-methyl-nitrosourea (MNU) and 1.3-bis (2-chloroethyl)-1-nitrosourea (BCNU) injected at maximal nonlethal single doses to mice bearing parent leukemia cells (L1210/0) and resistant to MNU and BCNU leukemia L1210 cells (L1210/MNU and L1210/BCNU), were studied. The MNU-induced production of single-strand breaks in L1210/0 and L1210/MNU cells was more conspicuous in newly replicated DNA than in those in preexisting DNA. A more fast repair of the damages in newly replicated DNA was detected in L1210/BCNU and especially in L1210/MNU leukemia cells as compared with L1210/0 cells. The data obtained suggest that there are prone errors in the repair of DNA template, since most of the single-strand breaks were revealed in the newly replicated DNA synthesized on the repaired DNA. The repair of DNA damages in L1210/BCNU and especially in L1210/MNU cells was accompanied by the activation of DNA-polymerases alpha and beta and poly(ADP-riboso)polymerase. Both DNA-polymerases--alpha and beta--were shown to be involved in repair of DNA damages induced by MNU and only DNA-polymerase beta was involved in the repair of damages induced by BCNU.     

A new 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay for testing macrophage cytotoxicity to L1210 and its drug-resistant cell lines in vitro

Summary Activated by interferon (IFN)(50 U/ml) and lipopolysaccharide (50 ng/ml), mouse peritoneal macrophages were cocultured with the L1210 parental cell line (L1210/PRT) and its Adriamycin-, cisplatin-resistant cell lines (L1210/ADM, L1210/CDDP) for 24 h at effector: target (E:T) ratios of 10:1, 5:1 and 2:1. The direct 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT)-cleavage assay, a new improved indirect MTT assay, and the colony-formation assay were used to quantify macrophage-mediated suppression of these non-adherent tumour targets. The results showed that the macrophages can produce formazan at a high level, which can interfere with the final results of a direct MTT assay. The new indirect MTT assay can avoid such interference because the effectors are separated from the targets before the assay is performed, so the real viability of the targets is reflected. An indirect MTT assay, as developed in this study, could be better than the direct assay for examining the suppressive effect of activated macrophages on non-adherent tumour cells in vitro. This study also revealed that all the L1210 cell lines can be suppressed significantly by the macrophages at E:T ratios of 10:1 and 5:1 while the two drug-resistant cell lines have lower survival rates at an E:T ratio of 10:1, indicating that they are more susceptible than their parental cell line.Supported by grants from the Ministry of Health and Welfare and the Ministry of Education, Science and Culture, Japan     

A mammalian cell variant in which 3-aminobenzamide does not potentiate the cytotoxicity of dimethyl sulphate

Variants of mouse leukaemia L1210 cells have been isolated in which cytotoxicity to dimethyl sulphate is not fully potentiated by ADP-ribosyl transferase inhibitor 3-aminobenzamide, as occurs in normal L1210 cells. These variants were selected after mutagenesis by growing the cells in dimethyl sulphate and 3-aminobenzamide. The characterisation of one of these variants is described. Variant 3 cells repair low doses of DNA damage in the presence of ADP-ribosyl transferase inhibitors. The Vmax of the ADP-ribosyl transferase enzyme in these cells is only increased 35% compared to normal wild-type L1210 cells. The basal DNA ligase I activity is increased 66% above wild-type whereas DNA ligase II activity appears to be unchanged. The most striking observation, however, is that the DNA ligase II activity is not increased after dimethyl sulphate treatment as occurs in wild-type L1210 cells. It seems that by increasing DNA ligase I levels these cells can survive DNA damage in the presence of 3-aminobenzamide. This variant (mutant) provides genetic evidence for our previously published hypothesis that (ADP-ribose)n biosynthesis is required for efficient DNA repair after DNA damage by monofunctional alkylating agents, because ADP-ribosyl transferase activity regulates DNA ligase activity. This variant is the first mammalian cell reported in which DNA ligase activity is altered, as far as we are aware. In yeast, a DNA ligase mutant has a cell division cycle (cdc) phenotype. Presumably, DNA ligase is essential for DNA synthesis, repair and recombination. The present variant provides further evidence that in mammalian cells, DNA ligase II activity is related to ADP-ribosyl transferase activity.