[Synthesis and anti-tumor properties of myelopeptide MP-1].

We have studied anti-tumor properties of bone marrow derived peptide Phe-Leu-Gly-Phe-Pro-Thr (MP-1) synthesized by classical methods. It was shown that MP-1 enhanced the effect ofcytostatic therapy of lymphatic leukemia P388 and increased latent growth period of P388 tumors implanted in irradiated mice. MP-1 also decreased metastasis of mouse breast adenocarcinoma Ca-755 after surgery.

Binding with serum components favorably affects cellular uptake of 111In-oligonucleotide in a leukemia cell line.

PURPOSE: The influence of serum components on the intracellular uptake of an 111In-oligonucleotide (ODN) against mdr1 mRNA was investigated in the murine leukemia cell line, P388/S, and its mdr1-overexpressed P388/R. METHODS: 111In-ODNs naked and vectorized with lipids were analyzed for binding with serum components using high-performance liquid chromatography (HPLC). 111In-ODN was incubated in albumin and transferrin solutions. 111In-DTPA and 111In-mononucleotide were incubated in serum. Degradation of naked 111In-ODN was detected in phosphate buffered saline (PBS) and serum containing endonuclease S1. Cellular uptakes of naked and vectorized 111In-ODN in the above cells were examined with and without fetal calf serum (FCS). RESULTS: Time-dependent binding of naked and vectorized 111In- ODN with serum components was observed throughout 24 hours. Transchelation of 111In to transferrin was not detected. HPLC profiles of 111In-DTPA and 111In-mononucleotide did not change in serum. Degradation of 111In-ODN by S1 was less remarkable in serum than in PBS. Specific accumulation of vectorized 111In-ODN in P388/R cells was achieved in culture with and without 10% FCS. CONCLUSIONS: This study verified the intense binding of ODN with serum components, leading to no inhibition on ODN intracellular specific uptake. Binding with serum components protects 111In-ODN from degradation by endonuclease and thus may facilitate ODN transmembrane delivery.

[Cisplatin and derivatives with radiation therapy: for what clinical use?]. / Sels de platine et radiothérapie: vingt ans de pratique.

Since its discovery by Rosenberg in 1965, cisplatin and its derivatives have appeared as the most important chemotherapeutic agents, particularly for their radiosensitizing properties and their clinical use with radiation. In spite of numerous preclinical and clinical studies, optimal schedules of platin and radiotherapy combination have to be defined. The first part of this overview will describe biological mechanisms of interaction between radiation therapy and platinum derivatives. The second part will report the major clinical impact of their association.

Radiobiological significance of beamline dependent proton energy distributions in a spread-out Bragg peak.

Similar target doses can be achieved with different mixed radiation fields, i.e., particle energy distributions, produced by a practical proton beam and a range modulator. The dose delivered in particle therapy can be described as the integral of fluence times the total mass stopping power over the particle energy distributions. We employed Monte Carlo simulations to explore the influence on the relative biological effectiveness (RBE) of the energy and the energy spread of the proton beam incident on a range modulator system. Using different beams, the conditions of beam delivery were adjusted so that similar spread out Bragg peak (SOBP) doses were delivered to a simulated water phantom. We calculated the RBE for inactivation of three different cell lines using the track structure model. The RBE depends on the details of the dose deposition and the biological characteristics of the irradiated tissue. Our calculations show that, for differing beam conditions, the corresponding differences in the total mass stopping power distributions are reflected in differences in the RBE. However, these differences are remarkable only at the very distal edge of the SOBP, for low doses, and/or for large differences in beam setup.

Biological effects of PEMF (pulsing electromagnetic field): an attempt to modify cell resistance to anticancer agents.

Pulsing Electromagnetic Field (PEMF) effects lead to a modification of the multidrug resistance (MDR) of cells in vitro and in vivo. The murine leukemic doxorubicin-resistant cell line, P388/Dx, subjected to PEMF irradiation in vitro, showed a significant difference in thymidine incorporation when the concentration of doxorubicin reached a level of 1 microgram/mL, which corresponds to the inhibition dose 50 (ID50). The human lymphoblastic leukemia vinblastine-resistant cell line, CEM/VLB100, also showed a significant modification under the same experimental conditions at the in vitro ID50 corresponding to a vinblastine concentration of 100 ng/mL. BDF1 mice transplanted with P388/Dx cells also had an increase in their life span when doxorubicin was injected intraperitoneally in fractionated doses, while being subjected to PEMF irradiation.

Synthesis of potential dual-acting radiation sensitizer antineoplastic agents: 2,2-dimethylphosphoraziridines containing 2-nitroimidazoles or other electron-affinic moieties.

In view of the in vivo demonstrated radiation-potentiating activities of several previously studied 2,2-dimethylphosphoraziridines, six new compounds incorporating the bis(2,2-dimethyl-1-aziridinyl)phosphinyl moiety, together with an electron-affinic group such as 2-nitroimidazole or nitrobenzyl, have been synthesized and tested (1) in vitro for ability to increase the effect of X-irradiation under hypoxic conditions on V-79 Chinese hamster lung fibroblast cells, (2) in vivo for antitumor activity in the absence of radiation against P388 leukemia in mice, and (3) in a preliminary experiment with compound 10 only, in combination with whole-body gamma-radiation, using the P388 leukemia mouse model for in vivo radiation-potentiating activity. The chemical-alkylating activities and hydrolytic behavior of these compounds, as well as their antitumor activities without radiation, were found to be comparable to those of other 2,2-dimethylphosphoraziridines, while their in vitro radiosensitizing activities were at low concentrations generally comparable to that of misonidazole, with compound 8 showing superior activity. At higher concentrations, only compound 10 was sufficiently soluble and nontoxic to the cells for evaluation in this assay. Thus, the bis(2,2-dimethyl-1-aziridinyl) phosphinyl moiety does not seem to have contributed to the hypoxic radiosensitizing activities (only to the cytotoxicities) of the electron-affinic moieties in this in vitro assay. In comparison, the prototype 2,2-dimethylphosphoraziridine, ethyl [bis(2,2-dimethyl-1-aziridinyl) phosphinyl]carbamate (AB-132), showed at nontoxic doses no radiosensitizing activity in this assay, and at cytotoxic doses increased the cell-killing effect of each given dose of X-radiation additively under both hypoxic and oxic conditions. Conversely, only the 2,2-dimethylphosphoraziridine moiety appeared to participate in the moderate "therapeutic radiation-potentiating" activity indicated by compound 10 in the in vivo experiment using the P388 leukemia model (on day 1), as the misonidazole standard was inactive in this nonhypoxic system. Clearly, the mechanism of the in vivo observed radiation-potentiating effect of AB-132 and other 2,2-dimethylphosphoraziridines is different from that of the hypoxic radiosensitizers, but the possible synergism between the two biologically active moieties of the new compounds could not be demonstrated with the experimental models so far employed.

Prevention of adverse effects of gamma-ray irradiation after metallothionein induction by bismuth subnitrate in mice.

The effect of preinduction of metallonthionein (MT) by bismuth subnitrate (BSN) on the adverse effects and antitumor activity of gamma-ray irradiation was investigated in mice. Preinduction of MT by oral administration of BSN significantly reduced the lethal effects and bone marrow injury caused by total body irradiation with gamma-rays. A significant increase in the MT concentration in bone marrow was observed in mice treated with BSN. In tumor-bearing mice, pretreatment with BSN did not compromise the antitumor activity of gamma-ray irradiation although bone marrow injury was remarkably suppressed. These results suggest that BSN pretreatment is an effective method for protection against side-effects in radiotherapy.

Effect of hyperthermia and X-irradiation on survival and occurrence of metastases in mice bearing P388 tumor.

Survival of P388 lymphoid tumor-bearing mice and the occurrence of metastasis was studied after combined modality treatment with hyperthermia and X-irradiation. P388 ascites tumor cells were treated at 42 degrees C or 43.5 degrees C for 1 hr in vitro and transplanted on B6D2F1 mice intraperitoneally (i.p.) or intramuscularly (i.m.). Hyperthermic treatment at 43.5 degrees C increased the median survival time (MST). Increased life-span (ILS) was found after i.p. transplantation (54%) and after i.m. transplantation (30%). During the life-span of tumor-bearing animals, significantly fewer metastases were observed in liver and spleen after hyperthermia and 5-10% metastasis occurred after transplantation of ascites tumor cells treated at 43.5 degrees C in vitro compared with 90% in the untreated control animals. The lower occurrence of metastasis could not be ascribed to the higher cell-killing effect of hyperthermia. When both modalities were combined the best tumor growth retardation effect was obtained when ascites tumor cells were treated at 43.5 degrees C for 1 hr before being transplanted i.m. and 1 day later locally X-irradiated with 6 Gy. In this case, 77% ILS was found demonstrating a synergistic effect of the two modalities. While X-irradiation alone did not change the occurrence of metastasis, after combined modality treatment it was as low as with hyperthermia alone (5-10%). In connection with the significantly lower occurrence of metastasis, the possible alterations of P388 tumor cell membrane and surface proteins induced by in vitro hyperthermic treatment are discussed.

Flow cytometric screening for selective toxicity to multidrug-resistant cells.

Mixed cultures originally containing doxorubicin [(DOX) NCS-123127]-sensitive leukemia P388 (P388/S) cells and DOX-resistant leukemia P388 (P388/R) cells were treated with drugs for 1 hour or with radiation, grown until the cell numbers had increased 250 times (8 cell doublings), and incubated with 2 micrograms daunorubicin (NCS-82151)/ml for 1 hour. The fluorescence of intracellular anthracycline measured on a flow cytometer was used as a marker to distinguish P388/S and P388/R cells. The proportions of low-fluorescent P388/R cells and high-fluorescent P388/S cells were determined from fluorescence histograms. Selective toxicity for multidrug-resistant P388/R cells was indicated by the decreased proportion of these cells in the mixed cultures. In untreated cultures grown from suspensions containing equal proportions of P388/R and P388/S cells, the mean proportion of P388/R cells after 4 days' growth was 34.4%. DOX eliminated P388/S cells from mixed cultures. Nitrogen mustard [(HN2) NCS-762] and x-rays were selectively toxic to P388/R cells. The selectivity of HN2 and x-rays was observed in a narrow dose range by the absence of selective inhibition at low doses, the decreased proportion of P388/R cells at moderate doses, and the killing of all cells in mixed cultures at high doses. Combination treatment of mixed cultures with DOX plus x-rays, or HN2 plus x-rays, produced complete inhibition of growth. Mixed cultures recovered after treatment with DOX plus HN2 contained only P388/R cells. Results obtained by colony-formation assay showed the same patterns of relative sensitivity for P388/R and P388/S cells as the data obtained by flow cytometry (FCM) selectivity assay. FCM analysis of mixed cultures detected selective toxicity for P388/R cells after treatments, characterized by approximately 1 log higher cell killing of P388/R cells than of P388/S cells. It was concluded that FCM analysis of mixed cultures provided a sensitive and reliable assay for fast screening of drugs for selective toxicity to multidrug-resistant cells.

Enhancement of radiation sensitivity by postradiation hypoxia.

We demonstrate that postradiation hypoxia during colony formation in vitro enhances radiation sensitivity when cells are irradiated in severely hypoxic states. The presence and magnitude of this phenomenon, if it occurs in vivo, raise questions about the importance of the oxygen effect in the radiation response of tumor cells in vivo and suggest that hypoxia may not be an important factor in the probability of tumor control following radiation therapy.

Mechanism of antitumor action of pyrimidinones in the treatment of B16 melanoma and P388 leukemia.

This study was undertaken in an attempt to understand the mechanism of antitumor action of pyrimidinones alone and in combination with cyclophosphamide (CY). Pyrimidinones such as 2-amino-5-bromo-6-(3-fluorophenyl)-4(3H)pyrimidinone (ABMFPP) were relatively nontoxic toward murine L1210 leukemia cell growth in vitro with the concentration of drug required for a 50% inhibition of cell growth being greater than 50 micrograms/ml. In contrast, ABMFPP showed anti-B16 melanoma activity in vivo which was sensitive to X-irradiation of the hosts. These results collectively suggest that pyrimidinones may act differently from conventional cytotoxic antitumor agents. Multiple i.p. injections of ABMFPP (125 mg/kg/injection) significantly augmented the cytotoxicity of both natural killer cells and macrophages in peritoneal exudates. The augmentation of both effector cell populations was delayed, but was more pronounced when animals received a dose of CY (100 mg/kg) prior to ABMFPP injections. The combination of CY and ABMFPP also showed a synergistic anti-P388 leukemia effect which appeared to be related to the initial reduction of the tumor burden by CY and the marked augmentation of the cytotoxicity of both natural killer cells and macrophages by ABMFPP. The antitumor activity of ABMFPP against B16 melanoma was almost completely eliminated when animals received a dose of 400 rads X-irradiation 5 days prior to tumor inoculation or a dose of 200 rads X-irradiation followed by several injections of anti-asialo monosialoganglioside antibody. The administration of anti-asialo monosialoganglioside alone also markedly reduced the anti-B16 melanoma activity of ABMFPP. The magnitude of reduction of the antitumor effect of ABMFPP by radiation and/or anti-asialo monosialoganglioside antibody directly correlated with the inhibition of the ABMFPP-mediated augmentation of immune responses. These results strongly suggest that the antitumor effect of ABMFPP alone or in combination with CY is at least in part mediated through its augmentation of natural killer cell and/or macrophage activities.

Thymosin alpha 1 restores NK-cell activity and prevents tumor progression in mice immunosuppressed by cytostatics or X-rays.

The effect of thymosin against tumor progression was examined in mice immunosuppressed by cytostatics or X-ray irradiation. When pretreated with cytostatic agents, such as 5-fluorouracil (5-FU) or BCNU, or by X-ray, and then inoculated with P388 or L1210 leukemias, mice died rapidly within a few days. In these systems, thymosin alpha 1 given concomitantly with the cytostatic agents or after X-irradiation prevented rapid death and extended survival, although the mice eventually died with leukemia like normal mice inoculated with cells of the same tumor. Rapid death in the 5-FU-treated mice was also prevented by adoptive transfer of spleen cells from the donor mice if these had been treated with 5-FU plus thymosin alpha 1, but not if they had received 5-FU alone. However, the restorative activity of the donor spleen cells was abrogated by treatment with anti-asialo GM1, but not by treatment with anti-Thy 1 or anti-mouse Ig serum, suggesting that the effector cells in the spleen are NK cells. In fact, thymosin alpha 1, when given concomitantly with 5-FU or after X-irradiation, maintained the NK activity of spleen, which was damaged by treatment with 5-FU or X-irradiation alone. The present study indicates that thymosin alpha 1 exerts a preventive activity against progression of leukemias at least in part through an effect on NK cells or their progenitor cells.