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.     

Synthesis,antineoplastic and cytotoxic activities of some mononuclear Ru(II) complexes

A series of mononuclear Ru(II) complexes of the type [Ru(S)₂(K)]²⁺, where S = 1,10-phenanthroline/2,2′-bipyridine and K = 4-OH-btsz, 4-CH₃-btsz, 3,4-di-OCH₃-btsz, 4-OH-binh, 4-CH₃-binh, 3,4-di-OCH₃-binh, were prepared and characterized by elemental analysis, FTIR, ¹H-NMR, and mass spectroscopy. The complexes displayed metal–ligand charge transfer (MLCT) transitions in the visible region. These ligands formed bidentate octahedral ruthenium complexes. The title complexes were evaluated for their in vivo anticancer activity against a transplantable murine tumor cell line, Ehrlisch’s ascites carcinoma (EAC), and in vitro cytotoxic activity against human cancer cell lines Molt 4/C₈ and CEM and murine tumor cell line L1210. The ruthenium complexes showed promising biological activity especially in decreasing tumor volume and viable ascites cell counts. Treatment with these complexes prolonged the life span of mice bearing EAC tumors by 10–52%. In vitro evaluation of these ruthenium complexes revealed cytotoxic activity from 0.21 to 24 μM against Molt 4/C₈, 0.16 to 19 μM aginst CEM, and 0.75 to 32 μM against L1210.     

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.     

Selected nuclear LINE elements with mitochondrial-DNA-like inserts are more plentiful and mobile in tumor than in normal tissue of mouse and rat

The nuclear DNA of normal and tumor mouse and rat tissue was examined for mitochondrial-DNA-like inserts by means of the Southern blot technique. The two probes were ³²P-labeled cloned mitochondrial DNA. KpnI, which doesn't cut either mitochondrial DNA, was one of the restriction enzymes, while the enzymes that fragment mitochondrial DNA were for mouse and rat PstI and BamHI, respectively. When KpnI alone was used in the procedure a nuclear LINE family whose elements had mitochondrial-DNA-like insertions was selected. Such elements were much more abundant in tumor than in normal tissue. The results with PstI alone and BamHI alone and each combined with KpnI indicated that there were mobile LINE elements with mitochondrial-DNA-like inserts in the nuclear genome of tumor. The mouse tissues were normal liver and a transplantable lymphoid leukemic ascites cell line L1210 that had been carried for 40 years. The rat tissues were normal liver and a hepatoma freshly induced by diethylnitrosoamine in order to minimize the role of 40 years of transplantation. Our unitary hypothesis for carcinogenesis of 1971, which suggested these experiments, has been augmented to include mobile nuclear elements with inserts of mitochondrial-DNA-like sequences. Such elements have been related to diseases of genetic predisposition such as breast cancer and Huntington's disease. J. Cell. Biochem. 68:100–109, 1998. © 1998 Wiley-Liss, Inc.     

Active oxidative decarboxylation of malate by mitochondria isolated from L-1210 ascites tumor cells

Mitochondria from L-1210 mouse ascites tumor show a very high rate of oxidation of L-malate in comparison with mitochondria from normal tissues. They were found to contain large amounts of malic enzyme (E.C.1.1.1.39) catalyzing oxidative decarboxylation of L-malate to pyruvate. Malic enzyme in extracts of tumor mitochondria requires Mn²⁺ or Mg²⁺, utilizes either NAD⁺ or NADP⁺ as electron acceptor, and shows positive cooperativity in binding of L-malate. These observations suggest that L-1210 tumor mitochondria actively convert excess tricarboxylate cycle intermediates and their precursors into pyruvate for further oxidation.     

In vitro and in vivo studies on antitumor effects of gossypol on human stomach adenocarcinoma (AGS) cell line and MNNG induced experimental gastric cancer

The present study has evaluated the chemopreventive effects of gossypol on N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis and on human gastric adenocarcinoma (AGS) cell line. Gossypol, C₃₀H₃₀O₈, is a polyphenolic compound that has anti proliferative effect and induces apoptosis in various cancer cells. The aim of this work was to delineate in vivo and in vitro anti-initiating mechanisms of orally administered gossypol in target (stomach) tissues and in human gastric adenocarcinoma (AGS) cell line. In vitro results prove that gossypol has potent cytotoxic effect and inhibit the proliferation of adenocarcinoma (AGS) cell line. In vivo results prove gossypol to be successful in prolonging the survival of MNNG induced cancer bearing animals and in delaying the onset of tumor in animals administrated with gossypol and MNNG simultaneously. Examination of the target (stomach) tissues in sacrificed experimental animals shows that administration of gossypol significantly reduces the level of tumor marker enzyme (carcino embryonic antigen) and pepsin. The level of Nucleic acid contents (DNA and RNA) significantly reduces, and the membrane damage of glycoprotein subsides, in the target tissues of cancer bearing animals, with the administration of gossypol. These data suggest that gossypol may create a beneficial effect in patients with gastric cancer.     

Inhibition of macromolecular synthesis in tumors by L-1-tosylamido-2-phenylethyl chloromethyl ketone.

L-1-tosylamido-2-phenylethyl chloromethyl ketone was observed to inhibit the incorporation of [³H] amino acids into protein and [³H] thymidine incorporation into DNA in Novikoff hepatoma ascites cells $\text{in vitro}$ Similar effects were seen with several Morris hepatomas and a transplanted colon tumor in rats, and were accompanied by decreased uptake of isotope into acid soluble tissue fractions. Under the same conditions, there was no significant inhibition in regenerating liver and there was an increased uptake of [³H] amino acids in the livers of normal and tumor bearing rats.     

Dose-Dependent Cytokinetic Changes Following 1-β-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-β-D-arabinofuranosylcytosine (Ara-C) and hydroxyurea (HU) were measured autoradiographically as a basis for optimizing drug schedules. Tumor bearing mice, 10⁶ cells day O, 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.

     

POST-MITOTIC AGE OF MONONUCLEAR CELLS MIGRATING INTO TA-3(St) SOLID TUMORS

Subcutaneous transplantation of 5 × 10⁶ TA-3(St) mammary carcinoma cells into A/J mice produced rapidly growing tumors that showed a substantial accumulation of lymphocytes, monocytes and macrophages. This must have resulted primarily from an influx of circulating cells, since none of the cell types exhibited any significant local proliferation as indicated by 1 hr ³H-TdR uptake. The post mitotic age of the various leukocyte types in circulation of normal and tumor bearing animals, and those appearing within the tumors, was evaluated by a repeated ³H-TdR labeling protocol designed to label newly formed leukocytes. Tumor transplantation (or injection of an equivalent volume of saline in control animals) was preceded by thirteen ³H-TdR injections (12.5 μCi every 8 hr) and followed by eight more injections at equivalent intervals. Animals were serially sacrificed at 5-14 days after tumor transplantation or saline injection. Total lymphocyte labeling in the blood during these intervals was higher in tumor bearing mice (approximately 25% between 5 and 12 days) than in the saline injected controls (approximately 20%), indicating that tumor transplantation resulted in an increase in the proportion of young lymphocytes in circulation. More significantly, a much higher labeling (57-60% at 5-12 days) was exhibited by lymphocytes isolated from the tumors, indicating selective migration (and/or retention) of newly formed lymphocytes within the tumor. This finding applied to lymphocytes in all size categories. Although blood monocyte labeling in the control and experimental animals was similar (e.g. 83% at day 5 and 52% at day 14), significantly higher labeling (e.g. 93% and 70% at the respective intervals) was exhibited by monocytes isolated from tumors. This suggested a preferential accumulation of young monocytes at the tumor site. An identical macrophage labeling pattern compared to that shown by monocytes within the tumor indicated a rapid monocyte-macrophage transition. Since these findings in the present strain-specific solid tumor are similar to those previously obtained in this laboratory in the strain-nonspecific Ehrlich ascites tumor, the phenomenon of selective localization of newly formed mono-nuclear leukocytes appears to be a general occurrence in transplanted murine tumors.     

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.     

Selective inhibition of thymidine transport at low doses of the alkylating agents triethyleneiminobenzoquinone (Trenimon)

The alkylating antitumor agent triethyleneiminobenzoquinone (Trenimon) causes a rapid decrease in the incorporation of labeled thymidine into the DNA of Yoshida or Ehrlich ascites tumor cells. The effect is expressed 4 h after administration of 6 × 10⁻⁸ moles/kg of the drug to mice bearing Yoshida ascites tumors or of 6 × 10⁻⁷ moles/kg to Ehrlich ascites tumor-bearing animals, respectively. The reduced incorporation of labeled thymidine which is observed under these conditions is not due to an inhibition of DNA synthesis. DNA synthesis was measured by an isotope dilution assay after pulse-labeling with ³H-thymidine and by monitoring the increase in the total amount of DNA of the cell populations. The data demonstrate that DNA synthesis is not affected during the first 8 h after exposure to the drug. This conclusion is supported by cell kinetic measurements which indicate that the alkylating agent does not interfere with the progression of cells into the S phase, but exerts a block at the G 2 stage of the cell cycle. The reduced incorporation of thymidine into DNA is explained by a decreased transport of the nucleoside into the cells.     

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.     

Stabilization of Membrane Bound Enzyme Profiles and Lipid Peroxidation by Withania Somnifera Along with Paclitaxel on Benzo(a)pyrene Induced Experimental Lung Cancer

The present study was aimed to evaluate the therapeutic effects of Withania somnifera along with paclitaxel on lung tumor induced by benzo(a)pyrene in male Swiss albino mice. The levels of ATPase enzymes and lipid peroxidation were evaluated in lung cancer bearing mice, in erythrocyte membrane and tissues. The extent of peroxidation was estimated by measuring the thiobarbituric acid-reactive substances. Simultaneously the activities of different ATPases (Na⁺/K⁺-ATPases, Mg²⁺-ATPases and Ca²⁺-ATPases) were determined. The alterations of these enzyme activities in membrane and tissues were indicative of the tumor formation caused by benzo(a)pyrene (50 mg/kg body weight, orally) in cancer bearing animals. The activities of these enzymes were reversed to near normal control values in animals treated with Withania somnifera (400 mg/kg b.wt, orally) along with paclitaxel (33 mg/kg b.wt, i.p). Treatment with Withania somnifera along with paclitaxel altered these damage mediated through free radicals, and the treatment displays the protective role of these drugs by inhibiting free radical mediated cellular damages. Over, based on the data providing a correlation Withania somnifera along with paclitaxel provide stabilization of membrane bound enzyme profiles and decreased lipid peroxidation against benzo(a)pyrene induced lung cancer in mice.     

IN VIVO EFFECTS OF 5-FLUOROURACIL AND FTORAFUR[1-(TETRAHYDROFURAN-2-YL)-5-FLUOROURACIL] ON MURINE MAMMARY TUMORS AND SMALL INTESTINE

The in vivo anti-tumour and toxic effects of ftorafur (FT) and 5-fluorouracil (FU) were studied in the C3H mouse. On a molar basis, FU was two to three times more potent than FT with respect to growth inhibition of murine mammary adenocarcinomas. However, FT produced less host toxicity than FU when both drugs were compared at dose levels which produced equivalent anti-tumor effects. The differences between FT and FU with respect to tumor growth inhibition and host toxicity were reflected in their ability to suppress deoxyuridine incorporation into tumor cell and intestinal DNA, respectively. Flow cytometry (FCM) studies indicated that FT and FU were capable of producing pertubations in the DNA distribution of tumour cells. Both drugs induced an initial accumulation of cells in S phase following their administration at equivalent anti-tumour dose levels. At later intervals, an apparent block of cell progression at the G₁/S boundary was observed. Drug-induced perturbations in the DNA distribution of tumour cells as detected by FCM correlated with results obtained by classical autoradiographic techniques using tritiated thymidine. Both procedures showed that tumor cells were capable of moving through S phase even in the presence of an apparently near complete inhibition of deoxyuridine incorporation into DNA. That such cells were, in fact, capable of synthesizing DNA at moderate rates was shown by their ability to incorporate ³²P into DNA. The possible relationship of these findings to the therapeutic and toxic activities of FT and FU is discussed.     

Antitumor effect of lysine-isopeptides

Isopeptides (ε-peptides) of lysine, with a given Mw and low polydispersity (10–400 units), were synthesized to study the relationship between their chemical structure and biological effect. The designed compounds were of high purity, low polydispersity and high stereochemical purity. The effect of the compounds was tested on a human erythroleukemia cell line (K-562) and on four transplantable mouse tumors (L1210 lymphoid leukemia, P38 macrophage derived tumor, Ehrlich ascites carcinoma, Lewis lung tumor /LLT/). In case of the L1210 and P388 tumors and the Ehrlich carcinoma, survival of the animals was used as an indicator of the effect. In case of the Lewis lung tumor, the number and size of metastases in the lung and/or liver of treated and untreated mice were used as indicators. The polymers of polymerisation degree 80–120 (Mw 10.2–15.4 KD) showed the strongest antiproliferative effect both on K562 cells and the tumors growing in vivo. This effect was manifest with a significantly higher survival rate as compared to the control (L1210, P38, Ehrlich ascites), furthermore, by a decrease in the number and size of liver and lung metastases (LLT).     

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.