Induction Of Chromosome Changes Research Articles

Concurrent Treatment with Pim Kinase Inhibitor Downregulates Alternative Non-Homologous End-Joining Repair and Decreases Genomic Instability in FLT3-ITD Cells Treated with Topoisomerase 2 Inhibitor Chemotherapy Drugs

Internal tandem duplication of fms -like tyrosine like kinase 3 (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML) cases, causing constitutive FLT3 signaling. While FLT3-ITD AML patients generally achieve remission following combination chemotherapy including cytarabine and a topoisomerase (topo) 2 inhibitor, they relapse rapidly, and FLT3-ITD AML cells frequently have new structural chromosome changes at relapse. Genomic instability in FLT3-ITD AML appears to result from increased generation of reactive oxygen species, which cause DNA double-strand breaks (DSBs), and upregulated DNA DSB repair by an error-prone alternative non-homologous end-joining (Alt-NHEJ) pathway.Pim-1 is an oncogenic serine/threonine kinase that is transcriptionally upregulated downstream of FLT3-ITD, contributes directly to the proliferative and anti-apoptotic effects of FLT3-ITD, and also phosphorylates and stabilizes FLT3, creating a positive feedback loop in FLT3-ITD cells. Pim kinase inhibitors are in clinical trials. We previously showed that concurrent treatment with a Pim kinase inhibitor enhances apoptosis induction by topo 2 inhibitors in FLT3-ITD cells. Here, we tested the effect of concurrent Pim kinase inhibitor treatment on Alt-NHEJ repair of topo 2 inhibitor-induced DNA DSBs and on genomic instability.Functional changes in DSB repair activities induced by treatment with the topo 2 inhibitor daunorubicin (DNR) in the absence and presence of the pan-Pim kinase inhibitor AZD1208 were studied in Ba/F3-ITD cells, with FLT3-ITD, using stably integrated green fluorescent protein (GFP)-based DSB repair reporter constructs DR-GFP, EJ5-GFP and EJ2-GFP, measuring homologous recombination (HR), classical (C-)NHEJ and Alt-NHEJ, respectively. DNR, which induced DNA DSBs, did not induce HR or C-NHEJ repair activity, but induced a 50% increase in Alt-NHEJ activity at 36 hours. Concurrent treatment with AZD1208, which increased DNR-induced DNA DSBs more than two-fold, completely abrogated the DNR-induced increase in Alt-NHEJ repair activity. Treatment of Ba/F3-ITD cells with the topo 2 inhibitors mitoxantrone (MXR) or etoposide (VP-16) also induced Alt-NHEJ repair activity, which was abrogated by concurrent treatment with AZD1208.We next studied mechanisms underlying Pim kinase inhibitor downregulation of Alt-NHEJ. The effects of DNR, MXR or VP-16 on cellular and nuclear expression of the Alt-NHEJ proteins poly (ADP-ribose) polymerase (PARP) 1, DNA polymerase θ, DNA ligase 3 and co-factor X-ray repair cross-complementing protein 1 (XRCC1) were measured in the FLT3-ITD cell lines Ba/F3-ITD, 32D-ITD, MV4-11 and MOLM-14 in the absence and presence of AZD1208. Cellular and nuclear expression of XRCC1 and DNA ligase 3, but not PARP or DNA polymerase θ, decreased in cells co-treated with AZD1208 and topo 2 inhibitors, compared to topo 2 inhibitors alone. XRCC1 was identified as a potential direct Pim-1 substrate by global profiling using the reverse in-gel kinase assay, and was confirmed as a robust Pim substrate in a reverse in-gel kinase assay. This provides a potential mechanistic link between Pim-1 kinase and ALT NHEJ.Alt-NHEJ repair is associated with genomic instability characterized by increased chromosome deletions, insertions and translocations. We studied induction of chromosome changes by topo 2 inhibitor treatment with and without Pim kinase inhibitor in FLT3-ITD cells. Ba/F3-ITD and 32D-ITD cells were incubated with DNR and/or AZD1208, or DMSO control, for 36 hours, then prepared for cytogenetic analysis.For each treatment, 20 consecutive metaphases were analyzed for induction of chromatid breaks and exchanges. No chromosome changes were seen in either cell line with DMSO or AZD1208 treatment. In Ba/F3-ITD cells, chromosome abnormalities were seen in 17 of 20 metaphases after DNR treatment, but only 8 of 20 after combination treatment (p=0.008). In 32D-ITD cells, chromosome abnormalities were seen in 8 of 20 metaphases with DNR, and 0 metaphases with combination (p=0.003).Thus concurrent treatment with Pim kinase inhibitor abrogates induction of Alt-NHEJ repair by topo 2 inhibitor treatment in FLT3-ITD cells and decreases the genomic instability that may contribute to rapid relapse of FLT3-ITD AML after chemotherapy. The data support further preclinical and clinical testing of Pim kinase inhibitors with chemotherapy in AML with FLT3-ITD. DisclosuresTron:AstraZeneca: Employment, Equity Ownership.

Effect of Phaulopsis falcisepala (Acanthaceae) Leaves and Stems on Mitotic Arrest and Induction of Chromosomal Changes in Meristematic Cells of Allium Cepa

Phaulopsis falcisepala is an herb found in the forest zone of West Africa. It is applied ethnomedicinally for treating wounds and cancer. This study investigated the chemical constituents, cytotoxicity, and antimitotic activity of P. falcisepala leaves and stems. Chemical constituents were evaluated using phytochemical methods. Cytotoxicity was investigated using brine shrimp lethality and Allium cepa root growth inhibition assays. Antimitotic activity and induction of chromosomal aberrations were assessed by cytological examination of treated A. cepa meristematic cells. Chemical investigation revealed the presence of diverse bioactive chemical groups, including alkaloids, terpenoids, tannins, flavonoids and saponins in various extracts of the leaves and stems of P. falcisepala. Aqueous and methanol extracts contained copious amounts of the phytochemicals while ethyl acetate and n-hexane extracts contained moderate to trace amounts. The extracts demonstrated toxicity against brine shrimps, with LC50 values in the range 13.1–52.2 µg/mL. The extracts significantly inhibited A. cepa root growth, with IC50 values in the range 63.91–87.4 µg/mL and 45.34– 81.15µg/mL for 24 and 48 h treatments. The extracts (IC50 values in the range 46.2–94.23 µg/mL and 30.79–75.81 µg/mL for 24 and 48 h treatments) produced significant antimitotic effect comparable to or greater than that of methotrexate (IC50 value >125 µg/mL). Cytological examinations revealed that the extracts induced mitotic arrest and chromosomal changes. This study reports for the first time the phytoconstituents and cytotoxic activity of P. falcisepala supporting its ethnomedicinal use and could serve as the basis for further pharmacological studies and isolation of bioactive principles.

Induction of chromosome changes by metal compounds in cultured CHO cells

The effect of 4 metal salts on the induction of chromosome aberrations and sister chromatid exchange (SCE) in cultured Chinese hamster ovary (CHO) cells was investigated. It was observed that CdCl 2, NiCl 2, CrO 3 and HgCl 2 were effective in causing various types of chromosome aberrations. The percentages of abnormalities increased with increasing doses of the metals used. These metal compounds also raised the SCE rates compared to 5-bromo-2'-deoxyuridine controls. The importance of rapid screening of potentially hazardous metals is described.

Single-particle irradiation of mammalian cells at the radiological research accelerator facility: induction of chromosomal changes

Ionizing radiations have been shown to be able to induce the death of mammalian cells and initiate mutagenic or carcinogenic change. While all three end points are related through chromosomal changes, the latter in particular is of profound concern to human populations. We have undertaken a series of studies wherein mammalian cells were irradiated with low fluences of charged particles (protons, deuterons, helium ions) of defined LET from 10 to about 200 keV per micrometer. Frequencies of induced chromosomal changes were related to fluence at each LET, such that the induction of chromosomal changes per cell per charged particle could be estimated. However, for human exposures from densely ionizing radiation, such as the alpha particles from radon daughters, effects are dominated by the traversal of cells by single particles. Conventional experiments inevitably result in cells being exposed to a distribution (Poisson) of particle traversals. As the effect is unlikely to be a linear function of the number of traversals, a preferred approach would be to irradiate cells with exactly one (or any known number) of particles. To this end we are developing a dedicated beam line (microbeam) on a 4.2 MV Van de Graaff accelerator such that individual particles will vertically traverse individual living mammalian cells positioned by a microscope-based imaging system under computer control. “Conventional” low-fluence and “single-particle” studies will be compared, allowing critical evaluations of the potential of individual high LET charged particles to initiate change. This will have particular relevance both to consideration of the human health risks of radon daughter alpha particles and of basic mechanisms of chromosome aberration formation.

Inhibition of carcinogen-induced chromosomal aberrations by an anticarcinogenic protease inhibitor.

It was hypothesized that chemicals- and radiation-induced carcinogenesis might require at least two specific chromosomal events that must coincide within a single target cell: (i) induction of chromosomal changes, possibly mutations, that are recessive and therefore latent in diploid somatic cells and (ii) aberrant mitotic segregation events that will convert the heterozygous cell, created by the first process, into a homozygous or hemizygous cell through chromosomal rearrangements. Hence, we tested the prediction that an inhibitor of induced carcinogenesis may inhibit one or both of these chromosomal events by studying the effects of antipain, a protease inhibitor and known inhibitor of carcinogenesis, on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis, chromosomal aberrations, sister chromatid exchanges, and cell killing in V79 Chinese hamster cells. We show that antipain inhibited MNNG-induced chromosomal exchanges and all other chromosomal aberrations exclusively. This results leads us to postulate that MNNG-induced DNA lesions cause chromosomal aberrations which arise through an antipain-sensitive cellular process, that some chromosomal rearrangement is a rate-limiting step in carcinogenesis, and that mutagenesis alone, if required, is not sufficient to accomplish carcinogenesis.

Induction of chromosome changes in Chinese hamster cells by exposure to asbestos fibres.

THE occurrence of pleural and peritoneal mesotheliomata is linked with the inhalation of asbestos fibres1,2. Experimentally, mesotheliomata have been induced by the intrapleural inoculation of asbestos fibres into rats3. These findings suggest that the incorporation of the fibres disturbs processes at the cellular level. The possibility that they induce chromosome abnormalities was indicated by V. Timbrell (personal communication) and we have investigated it further using cultured Chinese hamster cells.

CYTOKINETIC STUDY ON TOXIC ACTION OF SODIUM CYCLAMATE AND CYCLOHEXYLAMINE

In relation to induction of chromosome changes by cyclamates and cyclohexylamine, influence on cell renewal rate and inhibition of DNA synthesis of these chemicals were examined. Both sodium cyclamate and cyclohexylamine revealed to affect cell growth and to prolong S-phase of cell cycle in certain dose levels. The influence was stronger in case of cyclohexylamine treatment than sodium cyclamate in terms of dose-response relationship. The relationship between chromosome changes and mitotic inhibition is to be clarified by further investigations.