Effects of tubulozole on the microtubule system of cells in culture and in vivo.

Light and electron microscopic investigations on mammalian cells in vitro and in vivo showed that tubulozole-C (R 46 846), the cis-isomer of tubulozole, a new synthetic anticancer drug, interfered with the structure and function of microtubules in both interphase and mitotic cells. The activity of this compound in experimental tumor systems can thus be explained partly by a direct antimitotic effect and partly by the disintegration of the normal subcellular organization of the nondividing cells. At concentrations which affect the microtubule system, tubulozole-C arrested directional migration of transformed cells and malignant invasion in a three-dimensional organ culture system. Investigations in vivo show that malignant L1210 leukemia cells are more susceptible to the antimicrotubular effect of tubulozole-C than are the normal leukocytes of the host. The trans-isomer of tubulozole (tubulozole-T, R 48 265), which has no antitumor activity in vivo, did not affect the microtubule system of cells in vitro or their capacity for directional migration or for malignant invasion.

DL-2-Oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine. A sulfhydryl metabolite of levamisole that interacts with microtubules.

DL-2-Oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine (OMPI) a sulfhydryl metabolite of levamisole, unlike the parent compound, is shown to interfere with the morphological and functional integrity of microtubules in cultured cells at high concentrations (1.6-10(-4) M). Lower concentrations do not affect the cell morphology, viability or growth rate in any appreciable way. Both levamisole and OMPI, at low concentrations (10(-5)-10(-6) m), markedly enhance the antimicrotubular effect of mercaptoethanol. High concentrations of OMPI (+/- 10(-4) M) inhibit the self-assembly of microtubules in a cell free system. Low concentrations (+/- 10(-6) M) markedly enhance the polymerization rate of tubulin. Levamisole has no effect on tubulin polymerization. The effects of OMPI on microtubules in cells and in the polymerization system can be reversed by reduced glutathione, cysteine and dithiothreitol. The data indicate that OMPI interacts in a biphasic manner with microtubule formation probably through interaction with critical SH-groups on the tubulin molecule. It seems of interest to further investigate the hypothesis that the immunomodulating properties of levamisole are at least partially due to the formation of its metabolite (OMPI) which could enhance microtubule integrity and function in leukocytes.

The effects of methyl (5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl) carbamate, (R 17934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro.

Ultrastructural investigations on mammalian cells cultured in vitro show that R 17934, a new synthetic anticancer drug, interferes with the structure and function of microtubules, both in interphase and mitotic cells. The activity of this compound in a wide range of experimental tumor systems can thus be explained partly as a direct antimitotic effect and partly as the disintegration of the normal subcellular organization of the nondividing cells. Preliminary investigations in experimental animals show that malignant cells are more susceptible to the antimicrotubular effect of R 17934 than are the nonmalignant cells of the host.

A new culture model facilitating rapid quantitative testing of mitotic spindle inhibition in mammalian cells.

A new culture model, which facilitated both mass screening of potential anticancer drugs acting on microtubules and quantitative experiments with known "antitubulins," was found to have the following advantages: use of mammalian cells (either transformed or not), simplicity of the techniques (phase-contrast microscopy or simple microscopy after Giemsa staining), and ease with which it lent itself to quantification. The model was based on the uniform multimicronucleation response induced by antitubulins in MO cells. The specificity (towards antitubulins) of this response was ascertained by the use of many substances, including most of the known antitubulins and a number of nonrelated cytostatic or cytotoxic compounds. The uniformity of the response was established with the use of time-lapse observation of large numbers of cells and quantitative approaches. The results obtained in this model with the standard antitubulins (colchicine, vinblastine, vincristine) showed similar effects. The major difference between colchicine and the Vinca alkaloids was that colchicine was less reversible, which might be an indication of stronger intracellular binding. The Vinca alkaloids acted synergistically with colchicine when threshold subactive doses were combined, although it is known that they bind at a different site on tubulins. A number of substances that have been claimed or were suspected to interfere with microtubules were tested. The results showed that the following substances were indeed active with MO cells: colchicine, vinblastine, vincristine, podophyllotoxin, rotenone, griseofulvin, mercaptoethanol, benomyl, methyl benzimidazol-2-yl carbamate, and R 17934. Compounds that were inactive on these mammalian cells in culture included isopropyl carbanilate and melatonin, both of which have been shown to be active in other systems.