DNA Synthesis In Tumor Cells Research Articles

Doxorubicin conjugates of monoclonal antibodies to hepatoma-associated antigens.

A panel of six murine monoclonal antibodies against hepatocellular carcinoma-associated antigens, reactive with PLC/PRF/5 human hepatoma cells, was conjugated to Adriamycin (doxorubicin) via a dextran bridge. This library of antibodies includes three monoclonal antibodies against hepatitis B virus surface antigen, one anti-alpha-fetoprotein, and two other IgG2a antibodies against PLC/PRF/5 hepatoma-associated antigens. The use of dextran for conjugation of Adriamycin to antibodies enabled a 5- to 10-fold amplification of the number of drug molecules linked to antibody. Conjugation of Adriamycin to dextran caused an occasional reduction in the pharmacologic activity of dextran-Adriamycin in [3H]thymidine incorporation assays in hepatoma cells as compared to nonconjugated Adriamycin. This loss of anticellular activity was partially compensated for by conjugation of specific antibodies to the dextran-Adriamycin conjugate. Conjugated compounds completely retained their binding activity to purified hepatitis B virus surface antigen and alpha-fetoprotein fixed to a solid matrix as compared to binding of homologous nonconjugated antibodies. However, some reduction of the binding activity to intact hepatoma cells was observed in three of six conjugates. Binding activity to hepatoma cells and, as a consequence, suppression of tumor cell DNA synthesis by the various conjugates was enhanced as compared to the same effect in treated colorectal carcinoma cells that do not express the relevant hepatoma-associated proteins. Furthermore, two conjugates containing nonspecific antibodies did not bind to hepatoma cells and caused minimal suppression of DNA synthesis. These results suggest that this panel of monoclonal antibody-dextran-Adriamycin conjugates was effective in suppression of PLC/PRF/5 cell growth in vitro.

Enantiomers of 7-(2,3-epoxypropoxy)actinomycin D as dual-action DNA-acting antitumor agents.

Enantiomeric forms of (+/-)-EPA [racemic 7-(2,3-epoxypropoxy)actinomycin D] have been synthesized; these are (R)-(+)- and (S)-(-)-EPA, which are active against a range of actinomycin resistant and marginally responsive tumors. The R-(+) enantiomer is uniformly superior to the other forms in all the tumor lines tested. These enantiomers act by binding to DNA, both by intercalation and alkylation at the guanine base of DNA. They are superior to actinomycin D in their in vitro activity against mouse leukemias (L1210 and P388/ADR) and mouse melanoma B16. This superior activity is also evident against all the preceding mouse leukemias and against solid tumors B16 and C26 in vivo. In biochemical action, the enantiomers behave similarly and act primarily by inhibiting DNA synthesis in tumor cells; the only difference found was in their preference for sites in DNA bases during alkylation. The R-(+) enantiomer generates an adduct that is believed to be bonded to the N7-site of guanosine; conversely, the S-(-) isomer forms two adducts with DNA that are different from the preceding one by HPLC and are tentatively assigned O6-guanosine-substituted structures on the basis of their UV, CD, and other chemical behaviors.

Bisucaberin, a new siderophore, sensitizing tumor cells to macrophage-mediated cytolysis. I. Taxonomy of the producing organism, isolation and biological properties.

Alteromonas haloplanktis strain SB-1123 isolated from deep-sea mud produced a new siderophore, bisucaberin. Bisucaberin rendered tumor cells susceptible to cytolysis mediated by murine peritoneal macrophages which were elicited by Proteose peptone and not yet activated by lymphokine. Bisucaberin exerted its sensitizing activity by both the preincubation with tumor cells and the addition to co-culture of macrophages and tumor cells. The activity of bisucaberin was specifically inhibited by ferric ion. Bisucaberin showed direct cytostasis for tumor cells but did not cause cytolysis in the absence of macrophages. Cytostasis by bisucaberin was attributable to the specific inhibition of DNA synthesis in tumor cells.

Estimation of the potential antitumor activity of chemical compounds according to the inhibition of dna synthesis in tumor cells, spleen, and small intestine of C3HA mice with ascites hepatoma 22A

Estimation of the potential antitumor activity of chemical compounds according to the inhibition of dna synthesis in tumor cells, spleen, and small intestine of C3HA mice with ascites hepatoma 22A

Effect of preparations of glutamin(asparagin)ase from microorganisms on DNA synthesis in tumor cells

Effect of preparations of glutamin(asparagin)ase from microorganisms on DNA synthesis in tumor cells

Carbocyclic analogues of 5-fluorouracil nucleosides.

The carbocyclic analogues of 5-fluoro-2'-deoxyuridine (5-FdUrd, 1), 5-fluorouridine, and 5-fluoro-3 alpha-deoxyuridine were prepared by fluorination of the uracil nucleoside analogues with elemental fluorine. The 5-FdUrd analogue (C-5-F-2'-dUrd, 6) was enzymatically phosphorylated to the analogue of 5-FdUrd 5'-phosphate and inhibited the incorporation of 2'-deoxyuridine into DNA of murine colon 26 tumor cells and L-1210 cells in culture. Biochemical studies also indicated that C-5-F-2'-Urd (6) was a less potent inhibitor of DNA synthesis in tumor cells than was 5-FdUrd (1). C-5-F-2'-dUrd was cytotoxic (ED50 = 2.5 mcg/mL) to L-1210 cells in culture; the other two analogues were less cytotoxic. C-5-F-2'-dUrd was inactive--or, at best, borderline active--in tests against P-388 leukemia in vivo.

Prostaglandin analogs as inhibitors of tumor cell DNA synthesis.

AbstractAnalogs of the A and E series prostaglandins were screened in vitro for their chemotherapeutic potential against Lewis lung carcinoma and B16 amelanotic melanoma cells, since in vivo studies had demonstrated that PGA1 and an analog of PGE2 inhibited tumor growth. DNA synthesis by collagenase-dispersed tumor cells was evaluated at timed intervals after exposure to the 16, 16-dimethyl analogs of PGA1, PGA2, PGE1, and PGE2. In general, the PGA derivatives were more potent than the PGE analogs. However, the maximum effect was achieved with combined PGA and PGE treatment. These findings demonstrate that A and E series prostaglandin analogs inhibit DNA synthesis in two unrelated tumor types and thus may be potent inhibitors of tumor growth. These effects were not due to tumor cell cytotoxicity. These results suggest that these compounds may prove useful as cancer chemotherapeutic agents.

Action of cytosine arabinoside on human tumor cells isolated from malignant effusions: in vitro phosphorylation and inhibition of DNA synthesis.

Tumor cells were isolated from malignant effusions of three patients with disseminated solid tumors of different origin. Intracellular accumulation of nondiffusible cytosine arabinoside (ara-C) nucleotides was used to measure phosphorylation. Mouse leukemia L 1210 and L 1210/CA, and ara-C-resistant subline, were used as reference cells. Phosphorylation activity was similar in the cells from all three solid tumors and showed a linear increase with drug concentrations of 0.1--100 micograms/ml. In contrast to activity in L 1210 cells, the in vitro activity was not saturable at drug levels up to 100 micrograms/ml. Ara-C inhibited the incorporation of thymidine into DNA 84%--90% in the solid tumor cells at a concentration of 10 micrograms/ml. Higher drug concentrations did not result in further inhibition. In one patient, DNA synthesis of tumor cells isolated before and after intraperitoneal instillation of 1,000 mg ara-C was measured. The in vivo inhibition was found to correspond to the in vitro data. Solid tumor cells isolated from malignant effusion have no greatly reduced capacity for cellular formation of ara-C/nucleotides, but higher drug levels than achieved with conventional therapy are necessary for sufficient ara-C nucleotide synthesis.

Tumor Specific Immunity to Mouse Melanoma In Vitro

SummaryAn in vitro assay that measures the ability of mouse spleen cells to inhibit DNA synthesis (IDS) of syngeneic melanoma tumor cells was used in the evaluation of tumor immunity. The IDS assay was used in the measurement of both specific immunity mediated by spleen cells from tumor-bearing mice and nonspecific tumor cell growth inhibition mediated by normal spleen cells. Specific IDS was first demonstrable 2 weeks after injection of 106 B-16 tumor cells sub-cutaneously and persisted during advanced stages of disease. Changes in spleen cell IDS activity were correlated with changing mitogen reactivity during the course of tumor growth. A portion of the spleen cells mediating nonspecific IDS was removed by nylon wool filtration, but the effector cells exhibiting specific immunity were not removed by this procedure. Immune spleen cells did not need to synthesize DNA or divide to inhibit tumor cell DNA synthesis.

Effects on in vitro tumor growth of murine macrophages isolated from sarcoma lines differing in immunogenicity and metastasizing capacity

Macrophages were isolated from the strongly immunogenic, non-metastasizing FS6 sarcoma transplanted in syngeneic C57BL/6 mice and from a weakly immunogenic, spontaneously metastasizing line (mFS6) of the same tumor. Macrophages isolated from the FS6 sarcoma non-specifically inhibited growth and DNA synthesis of tumor cells in vitro. After culture FS6 macrophages rapidly lost their cytotoxic activity and became capable of non-specifically stimulating tumor growth in vitro. Macrophages obtained from the mFS6 tumor non-specifically enhanced the proliferative activity of tumor target cells and this capacity was maintained when the macrophages were cultured.

Antitumor Activity of Interferon Against Murine Osteogenic Sarcoma Cells In Vitro 2 3

Murine interferon inhibited the growth of a continuous line of osteogenic sarcoma (OGS) cells in tissue culture. Inhibition of tumor cell growth by interferon was demonstrated by: a) decreased colony formation in soft agar, b) suppression of clone formation in liquid medium, and c) reduction of tumor cell counts in monolayer cultures. This inhibition of cell growth was further documented by suppression of [3H]thymidine uptake by OGS cells exposed to interferon, which suggested inhibition of DNA synthesis of tumor cells. Exposure of tumor cells for 4 hours, 24 hours, and 2,3,4,6, and 8 days demonstrated greater activity with prolonged exposure to interferon. Inhibition of cell growth was significantly greater for OGS cells than for normal mouse embryo fibroblasts. Finally, the antitumor activity of the interferon preparation could be reversed by anti-interferon antibody.

In vitro sensitivity test for antineoplastic agents with an index of DNA synthesis of tumor cells

In vitro sensitivity test for antineoplastic agents with an index of DNA synthesis of tumor cells

Differential stimulation of murine lymphoma growth in vitro by normal and BCG-activated macrophages.

Peritoneal macrophages from mice infected with Bacille Calmette-Guérin (BCG) and from normal mice were examined for their effects in vitro on thymidine uptake by 10 murine lymphomas, a murine fibroblast line, and a guinea pig hepatoma. Only the murine fibroblast line showed growth inhibition in the presence of BCG macrophages. For the majority of tumors, normal macrophages were profoundly stimulatory to tumor cell DNA synthesis, while BCG macrophages were much less stimulatory, without being frankly inhibitory. The effect of 2-mercaptoethanol on tumor cell growth was also studied. All lymphomas stimulated to grow more rapidly in vitro by normal macrophages were stimulated to a similar degree by 2-mercaptoethanol.

Specific and Nonspecific Antitumor Immunity. I. Description of an In Vitro Assay Based on Inhibition of DNA Synthesis in Tumor Cells<xref ref-type="fn" rid="fn2">2</xref>

As in vitro assay of cell-mediated antitumor immunity based on the inhibition of tumor cell DNA synthesis (IDS) was devised. It was reasoned that both cytolytic and cytostatic antitumor effects could be measured in a quantitative yet generalized manner with this approach. By the use of microculture techniques and simplified methods for the determination of tritiated-thymidine incorporation by all cells in individual cultures, normal and immune effector (E) cell populations were compared in their ability to inhibit the DNA synthesis of a fixed initial number of various target tumor cells. Doubling dilutions of E cells were used to titrate the antitumor effects of normal and immune cells at many E/T (effector to target) ratios. Under conditions of alloimmunization, significant immunologically specific IDS effects could routinely be detected at an E/T ratio of less than 1 or 0.1:1, and under certain conditions at 1:100 or less. Results were highly reproducible with respect to the individual E cell donors, replicate cultures, and repeat experiments. The effects were proportional to visually determined cell destruction and independent of obvious culture artifacts. The IDS method was compared with the 51Cr release technique under various experimental conditions. The results demonstrated that decreases in E/T ratio and/or the addition of excess nonimmune cells to immune effector populations had a similar effect in both assays, which was to decrease the magnitude of the immune cell activity. Addition of excess normal cells reduced the activity of immune cells to a level below that of an equal number of immune cells tested at the same E/T ratio without added nonimmune cells. Both assays detected primarily a T lymphocyte-mediated lytic event when effectors generated by the described alloimmunizations were used. The IDS assay also detected a weak non-T-cell activity in anti-theta plus complement-treated alloimmune spleen. The possibility that this represented antibody-dependent cell-mediated cytotoxicity was raised by the finding that normal spleen ceels plus antitarget antibody had significant activity in the IDS system. The sensitivities of the two methods were compared and the potential of the IDS method was evaluated.

Uptake of 5-iododeoxyuridine as a measure of tumor growth and tumor inhibition.

The DNA synthesis of tumor cells in spleen, lung and liver of mice bearing spontaneous or transplanted lymphomas or Lewis lung carcinoma was determined by measuring the uptake of 5-iododeoxyuridine labeled with iodine-125 (125IUdR). An in vitro uptake method was also developed to assess 125IUdR uptake of peripheral blood of leukemic animals. A direct relationship was found between isotope uptake values and the extent of neoplastic growth. The 125IUdR uptake method was found to be applicable to determination of inhibition of tumor growth in drug-treated animals and in pre-sensitized allogeneic hosts and to measurement of regression in non-sensitized allogeneic mice.

Antitumor activities of bacterial leucine dehydrogenase and glutaminase a

Current observations on the inhibition of tumor growth by enzymes such as asparaginase [ 1,2], glutaminase [3] , arginase [4,5] and phenylalanine ammonia-lyase [6] , which catalyze the essentially irreversible degradation of amino acids, have stimulated the search for the antineoplastic activity of other microbial enzymes related to amino acid metabolism. The antitumor activities of a folate-cleaving bacterial enzyme, carboxypeptidase G, [7,8], and jack bean urease [9] , and the inhibition ofgrowth and DNA synthesis of tumor cells by ascorbic acid oxidase [lo] also have been recently reported. In the present communication we describe some studies on the antitumor activities of several bacterial enzymes, especially leucine dehydrogenase and isozyme A of glutaminase, which catalyze the reversible oxidative deamination of L-leucine and some other aliphatic amino acids in the presence of NAD [ 1 l] , and the deamidation of glutamine and asparagine [ 121, respectively.

Specific inhibition of tumor cell DNA synthesis in vitro by lymphocytes from peritoneal exudate of immunized syngeneic guinea pigs.

Tumor immunity to a transplantable diethylnitrosamine-induced hepatoma in inbred guinea pigs has been found to be immunologically specific and cell mediated. We have investigated this cellular immunity using a quantitative, reproducible, and simple assay based on the ability of leucocytes to inhibit the incorporation of tritiated thymidine (TdR(3)H) by tumor cells. Tumor cell suspensions were obtained from the ascites form or tissue culture monolayers of the hepatoma. Cells from the peritoneal exudate of immunized guinea pigs inhibited tritiated thymidine uptake by tumor target cells to a significantly greater degree than cells from the peritoneal exudate of unimmunized animals. Immune lymph node, peripheral blood, and spleen leucocytes were not inhibitory. The assay was sufficiently sensitive to detect relatively weak tumor immunity. The in vitro inhibition was correlated directly with the presence of delayed hypersensitivity and/or inhibition of tumor cell growth in local passive transfer studies. Irradiation of peritoneal exudate cells (3000 R) blocked their inhibitory effects on tumor cells. Fractionation of the peritoneal exudate cells by centrifugation in zones of bovine serum albumin of different density also revealed the lymphocytes to be responsible for the specific inhibitory effects whereas macrophages inhibited in an immunologically nonspecific fashion.