Olomoucine (2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine) has been recently described as a competitive inhibitor (ATP-binding site) of the cell cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase. The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle. The cellular effects of olomoucine were investigated in a large variety of plant and animal models. This compound inhibits the G1/S transition of unicellular algae (dinoflagellate and diatom). It blocks Fucus zygote cleavage and development of Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the development of Calanus copepod larvae. It reversibly inhibits the early cleavages of Caenorhabditis elegans embryos and those of ascidian embryos. Olomoucine inhibits the serotonin-induced prophase/metaphase transition of clam oocytes; furthermore, it triggers the the release of these oocytes from their meiotic metaphase I arrest, and induces nuclei reformation. Olomoucine slows down the prophase/metaphase transition in cleaving sea urchin embryos, but does not affect the duration of the metaphase/anaphase and anaphase/telophase transitions. It also inhibits the prophase/metaphase transition of starfish oocytes triggered by various agonists. Xenopus oocyte maturation, the in vivo and in vitro phosphorylation of elongation factor EF-1 are inhibited by olomoucine. Mouse oocyte maturation is delayed by this compound, whereas parthenogenetic release from metaphase II arrest is facilitated. Growth of a variety of human cell lines (rhabdomyosarcoma cell lines Rh1, Rh18, Rh28 and Rh30; MCF-7, KB-3-1 and their adriamycin-resistant counterparts; National Cancer Institute 60 human tumor cell lines comprising nine tumor types) is inhibited by olomoucine. Cell cycle parameter analysis of the non-small cell lung cancer cell line MR65 shows that olomoucine affects G1 and S phase transits. Olomoucine inhibits DNA synthesis in interleukin-2-stimulated T lymphocytes (CTLL-2 cells) and triggers a G1 arrest similar to interleukin-2 deprivation. Both cdc2 and cdk2 kinases (immunoprecipitated from nocodazole- and hydroxyurea-treated CTLL-2 cells, respectively) are inhibited by olomoucine. Both yeast and Drosophila embryos were insensitive to olomoucine. Taken together the results of this Noah's Ark approach show that olomoucine arrests cells both at the G1/S and the G2/M boundaries, consistent with the hypothesis of a prevalent effect on the cdk2 and cdc2 kinases, respectively.

EO9 is a novel and fully synthetic bioreductive alkylating indoloquinone. Although structurally-related to mitomycin C, EO9 exhibits a distinct preclinical antitumour profile and there are also differences in its biochemical activation. In this study, EO9 was found to demonstrate preferential cytotoxicity against solid tumours in vitro as compared to leukaemia cell lines both in the Corbett two-tumour assay and in the disease-oriented human tumour cell line panel of the U.S. National Cancer Institute. In the latter system activity was particularly apparent in colon, melanoma and central nervous system lines, together with some renal and non-small cell lung lines. Preferential cytotoxicity towards hypoxic versus aerobic EMT6 mouse mammary tumour cells was observed. In vivo, EO9 was inactive against the P388 murine leukaemia, while exerting significant antiproliferative effects against several murine and human solid tumours, including the generally resistant MAC mouse colon tumours and gastric, ovarian and breast xenografts. These results confirmed in vitro observations of preferential solid tumour activity. In animal toxicology studies, EO9 induced vascular congestion in the gastrointestinal tract, but no significant bone marrow toxicity. The LD10 value of EO9 after a single intravenous injection into mice was 9 mg/kg (27 mg/m2). A dose of one-tenth of the mouse equivalent LD10 (2.7 mg/m2), the recommended starting dose for clinical phase I studies, was found to be safe in rats. Considering its distinct mechanism of bioactivation as compared to mitomycin C, its preferential solid tumour activity, its excellent activity against hypoxic cells, and lack of significant bone marrow toxicity in animals studies, EO9 has been selected for clinical evaluation within the framework of the EORTC.

SummaryGemcitabine is a deoxycytidine (dCyd) analog with activity in leukemia and solid tumors, which requires phosphorylation by deoxycytidine kinase (dCK). Decreased membrane transport is a mechanism of resistance to gemcitabine. In order to facilitate gemcitabine uptake and prolong retention in the cell, a lipophilic pro-drug was synthesized (CP-4126), with an elaidic fatty acid esterified at the 5′position. CP-4126 was tested in cell lines resistant to cytarabine, another dCyd analog or gemcitabine. Activity of gemcitabine and the derivative was comparable in the parent cell lines, while in dCK deficient cells all compounds were inactive. However, inhibition of nucleoside transport increased the IC50 for gemcitabine up to 200-fold, but not for CP-4126, underlining the independence of a nucleoside transporter. For in vivo evaluation, nude mice bearing a human xenograft were treated intraperitoneally every third day for five doses at the maximal tolerated dose. In melanoma, sarcoma, lung, prostate, pancreatic and breast cancer xenografts, gemcitabine and CP-4126 were equally and highly effective; in four other xenografts moderately but equally active. In contrast to gemcitabine, CP-4126 could be administered orally, with a schedule and dose dependent toxicity and antitumor activity. In a colon cancer xenograft, antitumor activity of orally administered CP-4126 was equal to the intraperitoneally administered drug. In conclusion, CP-4126 is membrane transporter independent. Intraperitoneally administered CP-4126 was as effective as gemcitabine in several xenografts and CP-4126 is tolerated when orally administered. CP-4126 seems to be a promising new anticancer drug.

Purpose: KML001 (sodium metaarsenite) is an orally bioavailable arsenic compound that has entered phase I/II clinical trials in prostate cancer. In this study, we elucidated the mode of action of KML001and investigated its effects on telomerase and telomeres. Experimental Design: We compared telomere length to KML001cytotoxic activity in a panel of human solid tumor cell lines. Duration of exposure and concentrations of KML001 that affect telomerase and telomeres were evaluated in relation to established mechanisms of arsenite action such as reactive oxygen species^related DNA damage induction. Binding of KML001 to telomeres was assessed by matrix-assisted laser desorption/ionization mass spectrometry. Results: We established a significant inverse correlation (r 2 = 0.9) between telomere length and cytotoxicity. KML001exhibited activity in tumor cells with short telomeres at concentrations that can be achieved in serum of patients. We found that telomerase is not directly inhibited by KML001. Instead, KML001 specifically binds to telomeric sequences at a ratio of one molecule per three TTAGGG repeats leading to translocation of the telomerase catalytic subunit into the cytoplasm. In prostate cancer cells with short telomeres, KML001 caused telomere-associated DNA damage signaling as shown by g-H2AX induction and chromatin immunoprecipitation assays as well as a rapid telomere erosion shown by metaphase fluorescence in situ hybridization. These effects were not seen in a lung cancer cell line with long telomeres. Importantly, arsenification of telomeres preceded DNA lesions caused by reactive oxygen species production. Conclusions: Sodium metaarsenite is a telomere targeting agent and should be explored for the treatment of tumors with short telomeres.

Interruption of the afferent lymphatic vessels of the popliteal lymph node resulted in the disappearance of high endothelial venules (HEV) and immigrating lymphocytes within 3 weeks. HEV showed several characteristic morphological changes: the endothelial cells became flattened and less pyroninophilic, the chromatine became condensed and protein synthetizing and secretory cell organelles became scarce. At the same time the number of macrophages in the lymph node was severely reduced. Injection of sheep red blood cells into such lymph nodes, 6 weeks after operation, resulted in reappearance of HEV and immigrating lymphocytes, and development of many plasma cells and some germinal centres. Injection of lipopolysaccharide into the operated lymph nodes resulted in the appearance of many plasma cells and a few poorly developed germinal centres; HEV and immigrating lymphocytes, however, remained almost absent. The results show a relationship between the immigration of lymphocytes and the activity of the endothelial cells in the HEV. The activation of the latter may occur by mediators released by antigen-stimulated macrophages and T cells. Moreover, the morphological features of the HEV are independent of the presence of recirculating lymphocytes.