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  • To determine the time course of


    To determine the time course of ZD2767P+CPG2 DNA–DNA interstrand cross-link formation, HT29 and LS174T colorectal tumour NVP-ADW742 were exposed to the drug for 10, 30 or 60 min. The levels of DNA interstrand cross-links in HT29 or LS174T cells did not increase with exposure time, suggesting that ZD2767P+CPG2 forms DNA interstrand cross-links rapidly (<10 min). Rapid cross-link formation is consistent with reports that the cytotoxic species produced by ZD2767P+CPG2 has a very short half life (<2 min); a 1 min exposure having a similar IC50 (1.57 μM) to a 1 h exposure (0.34 μM) in LoVo cells [5]. Together, these data suggest that ZD2767P+CPG2 treatment generates a short-lived compound which produces cytotoxic DNA lesions very rapidly, consistent with the high chemical reactivity of ZD2767D. HN2 has also been shown to form maximal DNA interstrand cross-link levels rapidly (<1 h) [28], whereas melphalan and chlorambucil are reported to produce the maximal numbers of cross-links 4–8 h after the start of drug treatment 25, 28. The lack of any change in ZD2767+CPG2-induced cross-link levels over the 60 min period suggests that these adducts are relatively stable, although more extended post-treatment incubation would be needed to define the true stability, and possible repair, of these lesions. Together, the results of the alkaline elution and cell growth/cytotoxicity data strongly suggest that both the activity and DNA interstrand cross-linking produced by ZD2767P + CPG2 is due to bifunctionality of the active ZD2767D drug that is generated from ZD2767P. The monofunctional ZD2767D analogue is less potent and produces growth inhibition via a mechanism which does not involve DNA interstrand cross-linking, most probably the formation of DNA single-strand breaks, lesions observed following treatment with monofunctional HN2 analogues [23]. Comparison of the reported TP53 genotype of the cell lines used here with sensitivity to ZD2767P+CPG2 identified a weakly significant difference between the two groups; cell lines with wild-type TP53 being more sensitive. However, there were no differences in sensitivity to ZD2767P+CPG2, ZD2767P alone or chlorambucil in a pair of TP53 functional/non-functional HCT116 cell lines. In conclusion, these studies have demonstrated that ZD2767P+CPG2 generates a potent bifunctional alkylating agent which rapidly forms cytotoxic DNA interstrand cross-links. Comparisons with the classical nitrogen mustard chlorambucil in two panels of cell lines identified similar patterns of activity, suggesting common determinants of cellular sensitivity. The semi-quantitative relationship between the level of cross-links and drug sensitivity in the HT29, LS174T and NCI-H460 cell lines indicates that cross-link levels are a primary determinant of cellular sensitivity. In contrast, TP53 status did not clearly influence cellular sensitivity.
    Introduction During the times of the Green Empire, horticulturists used apple dwarfing rootstocks to reduce the size of the apple tree scion [1]. While dwarfing cultivation is being rapidly applied with new technologies and different apple varieties, the molecular mechanism underlying rootstock-induced scion dwarfing remains poorly understood. Cytokinin levels are lower in grafted scions of dwarfing rootstocks compared to vigorous rootstocks [2]. Applying 1-naphthaleneacetic acid (NAA) to the root does not reverse the root cytokinin levels in dwarfing rootstocks, but increases root auxin levels. Interestingly, when stem auxin transport is inhibited by NPA in vigorous rootstocks, root cytokinin level and shoot growth decline dramatically [3]. Taken together, these findings suggest that the dwarfing effect is caused by inherently poor root cytokinin biosynthesis in dwarfing rootstock. There are four known gene families in cytokinin biosynthetic pathway and one gene family in ctyokinin catabolism. Cytokinin biosynthesis begins with the enzyme isopentenyltransferase (IPT) catalyzing the addition of a prenyl NVP-ADW742 moiety from dimethylallyl diphosphate (DMAPP) to ATP/ADP, yielding N6-isopentenyladenine (iP) nucleotide [4], [5]. The Arabidopsis genome encodes nine IPT genes: IPT1 to IPT9. Aside from the pseudo gene IPT6, six IPT genes (IPT1, 3-5, 7-8) are involved in cytokinin biosynthesis, and IPT2 and IPT9 modify a subset of adenine bases on tRNAs [6]. The iP nucleotide made by IPT subsequently undergoes hydroxylation at the prenyl side chain by cytochrome P450 enzyme CYP735A, which converts iP to a trans-zeatin nucleotide [7]. Cytokinin nucleotides are activated by LONELY GUY (LOG), a phosphoribohydrolase, which converts trans-zeatin nucleotides into a free-base form by releasing a ribose 5′-monophosphate moiety from the cytokinin nucleoside 5′-monophosphate [8], [9]. Cytokinin oxidase (CKX) can irreversibly inactivate active by cleaving the N6-side chains from cytokinin [10], [11]. Interestingly, the susceptibility to CKX-mediated inactivation is quite different among the different cytokinins [12], [13], and iPs are more likely to be inactivated by CKX compared to other cytokinins [14].