Considerations for the genotoxicity assessment of middle size peptide drugs containing non-canonical amino acid residues.

BACKGROUND: Middle size peptides (MSPs) have emerged as a promising new pharmaceutical modality. We are seeking the best way to assess the non-clinical safety of MSPs. CONSIDERATION: The requirements for assessing the genotoxicity of pharmaceuticals differ between small molecule drugs and biotherapeutics. Genotoxicity tests are necessary for small molecule drugs but not for biotherapeutics. MSPs, however, share similarities with both small molecule drugs and biotherapeutics. Here, we describe important points to consider in assessing the genotoxicity of MSP drugs. The current standard of genotoxicity assessment for small molecules may not be entirely appropriate for MSP drugs. MSP drugs need genotoxicity assessment mostly according to the current standard of small molecule drugs. CONCLUSION: We propose a few modifications to the standard test battery of genotoxicity tests, specifically, the inclusion of an in vitro gene mutation test using mammalian cells, and exclusion of (Q)SAR assessment on MSP-related impurities.

Rapid in vitro assessment of the immunogenicity potential of engineered antibody therapeutics through detection of CD4+ T cell interleukin-2 secretion.

Therapeutic antibodies sometimes elicit anti-drug antibodies (ADAs) that can affect efficacy and safety. Engineered antibodies that contain artificial amino acid sequences are potentially highly immunogenic, but this is currently difficult to predict. Therefore, it is important to efficiently assess immunogenicity during the development of complex antibody-based formats. Here, we present an in vitro peripheral blood mononuclear cell-based assay that can be used to assess immunogenicity potential within 3 days. This method involves examining the frequency and function of interleukin (IL)-2-secreting CD4+ T cells induced by therapeutic antibodies. IL-2-secreting CD4+ T cells seem to be functionally relevant to the immunogenic potential due to their proliferative activity and the expression of several cytokines. The rates of the donors responding to low and high immunogenic proteins, mAb1, and keyhole limpet hemocyanin were 1.3% and 93.5%, respectively. Seven antibodies with known rates of immunogenicity (etanercept, emicizumab, abciximab, romosozumab, blosozumab, humanized anti-human A33 antibody, and bococizumab) induced responses in 1.9%, 3.8%, 6.4%, 10.0%, 29.2%, 43.8%, and 89.5% of donors, respectively. These data are comparable with ADA incidences in clinical settings. Our results show that this assay can contribute to the swift assessment and mechanistic understanding of the immunogenicity of therapeutic antibodies.

Preclinical in vitro evaluation of immune suppression induced by GYM329, Fc-engineered sweeping antibody.

Fc-engineering is commonly used to improve the therapeutic potency of antibody (Ab) treatments. Because FcγRIIb is the only inhibitory FcγR that contains an immunoreceptor tyrosine-based inhibition motif (ITIM), Fc-engineered Abs with enhanced binding affinity to FcγRIIb might provide immune suppression in clinical contexts. GYM329 is an anti-latent myostatin Fc-engineered Ab with increased affinity to FcγRIIb which is expected to improve muscle strength in patients with muscular disorders. Cross-linking of FcγRIIb by immune complex (IC) results in phosphorylation of ITIM to inhibit immune activation and apoptosis in B cells. We examined whether the IC of Fc-engineered Abs with enhanced binding affinity to FcγRIIb causes phosphorylation of ITIM or B cell apoptosis using GYM329 and its Fc variant Abs in human and cynomolgus-monkey (cyno) immune cells in vitro. IC of GYM329 with enhanced binding affinity to human FcγRIIb (×5) induced neither ITIM phosphorylation nor B cell apoptosis. As for GYM329, FcγRIIb should work as an endocytic receptor of small IC to sweep latent myostatin, so it is preferable that GYM329 induces neither ITIM phosphorylation nor B cell apoptosis to prevent immune suppression. In contrast, IC of myo-HuCy2b, the Ab with enhanced binding affinity to human FcγRIIb (×4), induced ITIM phosphorylation and B cell apoptosis. The result of the present study demonstrated that Fc-engineered Abs with similar binding affinity to FcγRIIb had different effects. Thus, it is important to also investigate FcγR-mediated immune functions other than binding to fully understand the biological effects of Fc-engineered Abs.

Priming treatment with T-cell redirecting bispecific antibody ERY974 reduced cytokine induction without losing cytotoxic activity in vitro by changing the chromatin state in T cells.

CD3 bispecific constructs are anticipated to become an important form of cancer immunotherapy, but they frequently cause cytokine release syndrome (CRS) that is difficult to manage in clinical contexts. A combination of intra-patient dose escalation and immunosuppressive treatment is widely used to mitigate CRS. Studies suggest that CRS after subsequent doses of CD3 bispecific constructs is less severe than after the priming dose, and that step-up dosing reduces cytokine levels in animals and humans. However, the mechanism underlying the reduced cytokine induction after priming treatment with CD3 bispecific constructs is unclear. To understand human T-cell activation and chromatin states after priming treatment with CD3 bispecific construct targeting CD3ɛ and glypican 3 (ERY974), we examined cytokine levels, cytokine mRNA expression, CD3ɛ expression, CD3-mediated signal transduction, T cell activation markers, cytotoxicity against target cells, and chromatin states in T cells after ERY974 priming treatment or negative control. The second ERY974 treatment decreased cytokines on Day 8, and ERY974 priming treatment changed the chromatin state in T cells. CD3ɛ expression, CD3-mediated signal transduction, T cell activation markers, and cytotoxicity were similar between the priming treatment with ERY974 and negative control. The present study suggests that chromatin state changes in T cells after the priming treatment was a pivotal factor in the mitigation of cytokine release after the second ERY974 treatment.

Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS.

BACKGROUND: Conflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies). RESULTS: Two of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay. CONCLUSIONS: The usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.

In vitro toxicological support to establish specification limit for anti-CD3 monospecific impurity in a bispecific T cell engager drug, ERY974.

An emerging structure for anti-tumor antibody drugs utilizes a bispecific antibody (BiAb) that recognizes a tumor surface antigen and CD3 on T cells. An impurity that commonly contaminates these BiAb products is an anti-CD3 monoclonal antibody (mAb). The most plausible cause of toxic activity by an anti-CD3 mAb is the induction of cytokines via T cell activation. In this in vitro study, we compared cytokine induction and T cell activation after treatment with an anti-glypican-3/CD3 BiAb (ERY974), anti-CD3 mAb impurity (aCD3), or ERY974 spiked with 5% aCD3. We found that contamination with up to 5% aCD3 did not affect cytokine release by ERY974. Cytokine levels induced by ERY974 in the presence of target cells were significantly higher than those induced by aCD3, but were very similar to those by the spiked treatment. The results supported the specification of a 5% limit for aCD3. OKT-3 had much higher activity to induce cytokines from peripheral blood mononuclear cells in an in vitro assay than aCD3. This suggests that specification limit should be decided for each type of anti-CD3 impurity that affects T cell-activating BiAb drug products. In vitro cytokine assays can provide useful information for determining these specification limits.

In vitro mammalian cell mutation assays based on transgenic reporters: A report of the International Workshop on Genotoxicity Testing (IWGT).

Chemical safety evaluations require assessment of genetic toxicity. Transgenic rodent (TGR) assays permit enumeration of mutations in chromosomally-integrated targets contained in shuttle vectors. In order to improve in vitro mutagenicity assessment, and to substantially reduce animal use, in vitro assays using transgenic reporters have been developed. These assays are based on cells derived from TGRs, or cells transfected with transgenic shuttle vectors containing a mutation target. As part of the 7th International Workshop on Genotoxicity Testing, an In Vitro Mammalian Cell Gene Mutation Assay working group reviewed all published information pertaining to in vitro transgene mutagenicity assays; the utility, advantages and disadvantages of the assays were evaluated and discussed. The review revealed that over 20 TGR-based in vitro assays have been used to assess the mutagenic activity of over 150 agents. Overall, the Working Group considered in vitro transgene mutagenicity assays pragmatic tools for the safety evaluation of new and existing substances. A formal SWOT (strengths, weaknesses, opportunities, threats) analysis revealed advantages including the use of established scoring protocols, avoidance of laborious clone isolation and enumeration, ability to use metabolically competent primary cells, ability to detect different types of genetic damage, large dynamic range, and complementarity to in vivo TGR endpoints. Disadvantages include lack of validation and little consistency in protocols, the use of specialised reagents, the time and effort required for mutant enumeration, the use of some cell lines that lack metabolic capacity, and the need for multiple assays to cover all mutational mechanisms. Several assays have been partially validated, indicating promising reliability, reproducibility and applicability domain. Once in vitro transgene mutagenicity assays have been more thoroughly validated, they are well placed to augment or replace existing in vitro mammalian cell mutagenicity assays, particularly in cases where the in vivo TGR mutation assay is intended for follow-up.

Daily ascending dosing in cynomolgus monkeys to mitigate cytokine release syndrome induced by ERY22, surrogate for T-cell redirecting bispecific antibody ERY974 for cancer immunotherapy.

CD3 bispecific constructs show promising therapeutic potential as anti-tumor antibodies, but it has concurrently been difficult to manage cytokine release syndrome (CRS) in clinical use. Currently, the most effective measure for reducing CRS is considered a combination of intra-patient/animal dose escalation and corticosteroid premedication. To examine how effectively an intra-animal ascending dose regimen without premedication would mitigate CRS, we compared plasma cytokine levels in two groups of cynomolgus monkeys; one group was given a single dose, and the other a three-fold daily ascending dose of a CD3 bispecific construct that targets and cross-reacts with both glypican 3 and CD3 (ERY22). Ascending doses up to 1000 µg/kg of ERY22 dramatically reduced the peak cytokine levels of IL-6, TNF-α, and IFN-γ, IL-2 as well the clinical severity of CRS compared with a single dose of 1000 µg/kg. Peak cytokine levels following the single and ascending doses were 60,095 pg/mL and 1221 pg/mL for IL-6; 353 pg/mL and 14 pg/mL for TNF-α; 123 pg/mL and 16 pg/mL for IFN-γ; and 2219 pg/mL and 42 pg/mL for IL-2. The tolerance acquired with daily ascending doses up to 1000 µg/kg remained in effect for the following weekly doses of 1000 µg/kg.

In vitro human helper T-cell assay to screen antibody drug candidates for immunogenicity.

Monoclonal antibody (mAb) drugs offer a number of valuable treatments. Many newly developed mAb drugs include artificial modification of amino acid sequences from human origin, which may cause higher immunogenicity to induce anti-drug antibodies (ADA). If the immunogenicity of a new candidate can be understood in the nonclinical phase, clinical studies will be safer and the success rate of development improved. Empirically, in vitro immunogenicity assays with human cells have proved to be sufficiently sensitive to nonhuman proteins, but not to human/humanized mAb. To detect the weaker immunogenicity of human-based mAb, a more sensitive biomarker for in vitro assays is needed. The in vitro study here developed a proliferation assay (TH cell assay) using flow cytometry analysis that can detect a slight increase in proliferating TH cells. Samples from 218 donors treated with a low-immunogenic drug (etanercept) were measured to determine a positive threshold level. With this threshold, positive donor percentages among PBMC after treatment with higher-immunogenicity mAb drugs were noted, that is, 39.5% with humanized anti-human A33 antibody (hA33), 27.3% with abciximab, 25.9% with adalimumab, and 14.8% with infliximab. Biotherapeutics with low immunogenicity yielded values of 0% for basiliximab and 3.7% for etanercept. These data showed a good comparability with previously reported incidences of clinical ADA with the evaluated drugs. Calculations based on the data here showed that a TH cell assay with 40 donors could provide statistically significant differences when comparing low- (etanercept) versus highly immunogenic mAb (except for infliximab). Based on the outcomes here, for screening purposes, a practical cutoff point of 3/20 positives with 20 donors was proposed to alert immunogenicity of mAb drug candidates.

High-content imaging analyses of γH2AX-foci and micronuclei in TK6 cells elucidated genotoxicity of chemicals and their clastogenic/aneugenic mode of action.

BACKGROUND: The in vitro micronucleus (MN) test is an important component of a genotoxicity test battery that evaluates chemicals. Although the standard method of manually scoring micronucleated (MNed) cells by microscope is a reliable and standard method, it is laborious and time-consuming. A high-throughput assay system for detecting MN cells automatically has long been desired in the fields of pharmaceutical development or environmental risk monitoring. Although the MN test per se cannot clarify whether the mode of MN induction is aneugenic or clastogenic, this clarification may well be made possible by combining the MN test with an evaluation of γH2AX, a sensitive marker of DNA double strand breaks (DSB). In the present study, we aimed to establish a high-content (HC) imaging assay that automatically detects micronuclei (MNi) and simultaneously measures γH2AX foci in human lymphoblastoid TK6 cells. RESULTS: TK6 cells were fixed on the bottom of each well in 96-well plates hypotonically, which spreads the cells thinly to detach MNi from the primary nuclei. Then, the number of MNi and immunocytochemically-stained γH2AX foci were measured using an imaging analyzer. The system correctly judged 4 non-genotoxins and 13 genotoxins, which included 9 clastogens and 4 aneugens representing various genotoxic mechanisms, such as DNA alkylation, cross-linking, topoisomerase inhibition, and microtubule disruption. Furthermore, all the clastogens induced both γH2AX foci and MNi, while the aneugens induced only MNi, not γH2AX foci; therefore, the HC imaging assay clearly discriminated the aneugens from the clastogens. Additionally, the test system could feasibly analyze cell cycle, to add information about a chemical's mode of action. CONCLUSIONS: A HC imaging assay to detect γH2AX foci and MNi in TK6 cells was established, and the assay provided information on the aneugenic/clastogenic mode of action.

Different players generate positive responses in two in vitro cytokine assay formats with aqueous and immobilized TGN1412 analog.

To detect potential risk of severe cytokine release syndrome, in vitro assay formats with human cells have been developed. The two major testing platforms are a combination of whole blood with aqueous-phase test articles (whole blood cytokine assay, WBCA) and peripheral blood mononuclear cells with solid-phase articles (PBMC assay). Significant induction of cytokines was seen in both assays after treatment with a widely used control agent, TGN1412 or its analog CD28SA, but the WBCA cytokine profile differed from what was expected from clinical experience. In the WBCA, potential risk of CD28SA was detected by elevation of IL-8 whereas IL-2, a key cytokine after stimulation of CD28, was not induced in approximately 40% of donor samples. Therefore, further mechanistic understanding of the different responses in the in vitro assay was needed. In this study of donor samples treated with CD28SA, we compared the induction of cytokines and identified the cytokine-producing cells in the two assays. IL-2 was markedly elevated in all the donors in the PBMC assay but only in 1 of 3 donors in the WBCA. IL-8, the most sensitive biomarker in the WBCA, was produced by monocytes and granulocytes. T cells, the most relevant player in the PBMC assay with CD28SA, did not contribute to the positive response seen in two donors in the WBCA, which suggests that different players caused the positive cytokine responses to CD28SA in the two assays.

Advantages of evaluating γH2AX induction in non-clinical drug development.

γH2AX, the phosphorylated form of a histone variant H2AX at Ser 139, is already widely used as a biomarker to research the fundamental biology of DNA damage and repair and to assess the risk of environmental chemicals, pollutants, radiation, and so on. It is also beginning to be used in the early non-clinical stage of pharmaceutical drug development as an in vitro tool for screening and for mechanistic studies on genotoxicity. Here, we review the available information on γH2AX-based test systems that can be used to develop drugs and present our own experience of practically applying these systems during the non-clinical phase of drug development. Furthermore, the potential application of γH2AX as a tool for in vivo non-clinical safety studies is also discussed.

Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene-induced genotoxicity in vivo.

DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc.

Giemsa-stained pseudo-micronuclei in rat skin treated with vitamin D3 analog, pefcalcitol.

BACKGROUND: Pefcalcitol, an analog of vitamin D3 (VD3), is an anti-psoriatic drug candidate that is designed to achieve much higher pharmacological effects, such as keratinocyte differentiation, than those of VD3, with fewer side effects. Genotoxicity of the compound was evaluated in a rat skin micronucleus (MN) test. RESULTS: In the rat skin MN test, pefcalcitol showed positive when specimens were stained with Giemsa, whereas neither an in vitro chromosome aberration test in CHL cells nor an in vivo bone marrow MN test in rats indicated clastogenicity. To elucidate the causes of the discrepancy, the MN specimens were re-stained with acridine orange (AO), a fluorescent dye specific to nucleic acid, and the in vivo clastogenicity of the compound in rat skin was re-evaluated. The MN-like granules that had been stained by Giemsa were not stained by AO, and AO-stained specimens indicated that pefcalcitol did not increase the frequency of micronucleated (MNed) cells. Histopathological evaluation suggested that the MN-like granules in the epidermis were keratohyalin granules contained in keratinocytes, which had highly proliferated after treatment with pefcalcitol. CONCLUSIONS: Pefcalcitol was concluded to be negative in the rat skin MN test. The present study demonstrated that Giemsa staining gave a misleading positive result in the skin MN test, because Giemsa stained keratohyalin granules.

Chromosomal aberrations, clastogens vs aneugens.

Current anticancer therapy may be one of the most important exogenous sources of exposure to genotoxic agents in US, Japan, and Europe, where approximately 40-55 percent of the population is diagnosed with cancer at a certain point in their life. This review focuses on recent efforts to integrate a novel biomarker, gamma-H2AX, into anticancer drug screening to classify the mode of action (MoA) for genotoxic outcome into clastogenicity and aneugenicity, a distinction that has considerable impact on risk assessment and control strategy. The emerging biomarker gamma-H2AX is applicable to high throughput assay platforms and is therefore changing in vitro mammalian genotoxicity screening from traditional positive/negative selection to MoA elucidation. Because gamma-H2AX is not only a sensitive biomarker for DNA double strand break but is also induced by apoptosis, the key for successful screening is using additional biomarkers of caspase-3 and/or phosphorylated histone H3 to discriminate between relevant and irrelevant elevation of gamma-H2AX. Establishment of a standard methodology and a consensus threshold for its positive criteria will further support the application of gamma-H2AX to drug screening.

Is an in vitro whole blood cytokine assay useful to detect the potential risk of severe infusion reaction of monoclonal antibody pharmaceuticals?

After the life-threatening cytokine release syndrome (CRS) occurred in the clinical study of the anti-CD28 monoclonal antibody (mAb) TGN1412, in vitro cytokine release assays using human blood cells have been proposed for non-clinical evaluation of the potential risk of CRS. Two basic assay formats are frequently used: human peripheral blood mononuclear cells (PBMC) with immobilized mAbs, and whole blood with aqueous mAbs. However, the suitability of the whole blood cytokine assay (WBCA) has been questioned, because an unrealistically large sample size would be required to detect the potential risk of CRS induced by TGN1412, which has low sensitivity. We performed a WBCA using peripheral blood obtained from 68 healthy volunteers to compare two high risk mAbs, the TGN1412 analogue anti-CD28 superagonistic mAb (CD28SA) and the FcγR-mediated alemutuzumab, with a low risk mAb, panitumumab. Based on the cytokine measurements in this study, the sample size required to detect a statistically significant increase in cytokines with 90% power and 5% significance was determined to be n = 9 for CD28SA and n = 5 for alemtuzumab. The most sensitive marker was IL-8. The results suggest that WBCA is a practical test design that can warn of the potential risk of FcγR-mediated alemtuzumab and T-cell activating CD28SA but, because there was apparently a lower response to CD28SA, it cannot be used as a risk-ranking tool. WBCA is suggested to be a helpful tool for identifying potential FcγR-mediated hazards, but further mechanistic understanding of the response to CD28SA is necessary before applying it to T cell-stimulating mAbs.

Integrated approach to testing and assessment for predicting rodent genotoxic carcinogenicity.

We investigated the performance of an integrated approach to testing and assessment (IATA), designed to cover different genotoxic mechanisms causing cancer and to replicate measured carcinogenicity data included in a new consolidated database. Genotoxic carcinogenicity was predicted based on positive results from at least two genotoxicity tests: one in vitro and one in vivo (which were associated with mutagenicity categories according to the Globally Harmonized System classification). Substances belonging to double positives mutagenicity categories were assigned to be genotoxic carcinogens. In turn, substances that were positive only in a single mutagenicity test were assigned to be mutagens. Chemicals not classified by the selected genotoxicity endpoints were assigned to be negative genotoxic carcinogens and subsequently evaluated for their capability to elicit non-genotoxic carcinogenicity. However, non-genotoxic carcinogenicity mechanisms were not currently included in the developed IATA. The IATA is docked to the OECD Toolbox and uses measured data for different genotoxicity endpoints when available. Alternatively, the system automatically provides predictions by SAR genotoxicity models using the OASIS Tissue Metabolism Simulator platform. When the developed IATA was tested against the consolidated database, its performance was found to be high, with sensitivity of 74% and specificity of 83%, when measured carcinogenicity data were used along with predictions falling within the models' applicability domains. Performance of the IATA would be slightly changed to a sensitivity of 80% and specificity of 72% when the evaluation by non-genotoxic carcinogenicity mechanisms was taken into account. Copyright © 2016 John Wiley & Sons, Ltd.

In vivo evidence that DNA polymerase kappa is responsible for error-free bypass across DNA cross-links induced by mitomycin C.

Translesion DNA synthesis (TLS) is an important pathway that avoids genotoxicity induced by endogenous and exogenous agents. DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in TLS but its protective roles against DNA damage in vivo are still unclear. To better understand these roles, we have established knock-in mice that express catalytically-inactive Polk and crossbred them with gpt delta mice, which possess reporter genes for mutations. The resulting mice (inactivated Polk KI mice) were exposed to mitomycin C (MMC), and the frequency of point mutations, micronucleus formation in peripheral erythrocytes, and γH2AX induction in the bone marrow was determined. The inactivated Polk KI mice exhibited significantly higher frequency of mutations at CpG and GpG sites, micronucleated cells, and γH2AX foci-positive cells than did the Polk wild-type (Polk(+)) mice. Recovery from MMC-induced DNA damage, which was evaluated by γH2AX induction, was retarded in embryonic fibroblasts from the knock-in mice when compared to those from the Polk(+) mice. These results suggest that Polk mediates TLS, which suppresses point mutations and DNA double-strand breaks caused by intra- and interstrand cross-links induced by MMC treatment. The established knock-in mice are extremely useful to elucidate the in vivo roles of the catalytic activity of Polk in suppressing DNA damage that was induced by a variety of genotoxic stresses.

The predominant role of apoptosis in γH2AX formation induced by aneugens is useful for distinguishing aneugens from clastogens.

The phosphorylated form of the histone protein H2AX, called γH2AX, is recognized as a useful biomarker not only for DNA double-strand breaks but also for a wide range of other DNA damage. An increasing number of publications propose γH2AX to be measured when determining genotoxicity, phototoxicity, and the effectiveness of cancer therapy. Because γH2AX is also generated by apoptosis, a γH2AX-assay might assess genotoxic risk incorrectly. The aim of this study was to elucidate the influence of apoptosis on measurements of γH2AX by flow cytometry, with the clastogens mitomycin C (MMC) and etoposide (ETP), and the aneugens vinblastine (VB) and paclitaxel (PT), which do not react directly with DNA. TK6 human lymphoblastoid cells were treated with the clastogens and the aneugens, stained for the apoptotic biomarker caspase-3 and for γH2AX, and then analyzed by flow cytometry. All the test compounds caused a dose-dependent increase of γH2AX-positive (γH2AX+) cells. The γH2AX+ cell population included both caspase-3-positive (γH2AX+/caspase-3+) and caspase-3-negative (γH2AX+/caspase-3-) cells. The increase in γH2AX+ cells after treatment with the aneugens corresponded to the increase in caspase-3+ cells. The increase in γH2AX+/caspase-3- cells after treatment with the clastogens was significant, but there was only a slight increase after treatment with the aneugens. This reflects the fact that the apoptotic pathway of a clastogen starts from DNA damage, whereas that of an aneugen starts from cell-cycle arrest in the M-phase. Therefore, the two pathways contribute differently to apoptosis. Double staining for γH2AX and caspase-3 provided helpful information for the different mechanistic effects of aneugens and clastogens that induce γH2AX.

HSP90 inhibitor CH5164840 induces micronuclei in TK6 cells via an aneugenic mechanism.

Heat-shock protein 90 (HSP90) is a promising druggable target for therapy of conditions including cancer, renal disease, and chronic neurodegenerative diseases. Despite the possible beneficial effects of HSP90 inhibitors, some of these agents present a genotoxicity liability. We have examined the mode of action of micronucleus formation in TK6 cells by a novel and highly specific HSP90 inhibitor, CH5164840, by means of an in vitro micronucleus test with fluorescence in situ hybridization (FISH), γH2AX staining to detect DNA damage, and microscopic observation of chromosomal alignment in mitotic cells. The percentage of centromere-positive micronuclei induced by CH5164840 (FISH analysis) was significant, but the percentage of centromere-negative ones was not, suggesting that induction of micronuclei was due to a mechanism of aneugenicity rather than DNA reactivity. This conclusion was further supported by the result of co-staining γH2AX and the apoptosis marker caspase-3; the predominant elevation of apoptotic γH2AX rather than non-apoptotic γH2AX indicated little involvement of DNA-reactivity mechanisms. Microscopic observation revealed asymmetric spindle microtubules and chromosomal misalignment of metaphase cells. These data indicated that CH5164840 causes spindle dysfunction that induces micronuclei. The risk/benefit ratio must be considered in the development of HSP90 inhibitors.