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Immunohistochemical Analysis of Acetylation, Proliferation, Mitosis, and Apoptosis in Tumor Xenografts Following Administration of a Histone Deacetylase Inhibitor—A Pilot Study
1 School of Biosciences, Birmingham University, Edgbaston, Birmingham, United Kingdom Correspondence: Address correspondence to: Tijana Mitiæ, Centre for Cardiovascular Science, Queen’s Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, United Kingdom; e-mail:t.mitic{at}sms.ed.ac.uk
Histone acetyltransferases and histone deacetylases are protein-modifying enzymes involved in addition and removal of acetyl groups on histone proteins, respectively. These molecules play a pivotal role in cellular functions such as chromosome remodelling, gene transcription and cell proliferation. Histone deacetylase inhibitors (HDIs) have been shown to cause cell cycle arrest, cellular differentiation and inhibition of cell proliferation in tumor cells in vitro and in vivo. Their potential use for cancer therapy is currently under evaluation in clinical trials. A pilot study was performed to immunohistochemically evaluate the effects of a HDI, "Compound 1," on acetylation, proliferation, mitosis, and apoptosis in tumor xenografts (Calu-6, SW 620, Colo 205, and LoVo) in nude mice, at 6, 24, and 48 hours, following a single oral dose. Qualitative immunohistochemistry and computer-assisted image analysis demonstrated an increase in acetylation in all xenografts. Immunohistochemical analysis of acetylation in skin showed increased acetylation at 6 hours after HDI administration. In addition, image analysis showed a decrease in mitosis and an increase in metaphase mitotic figures in the SW 620 xenograft. These two findings were consistent with a G1/S cell cycle phase arrest. Increased apoptosis of SW 620 and LoVo xenografts was also observed following treatment.
Key Words: Acetylation • proliferation • apoptosis • xenografts • animal model • pharmacodynamics Abbreviations: IHC, immunohistochemistry • HDAC, histone deacetylase • HDI, histone deacetylase inhibitor • HAT, histone acetyltransferase • AcH3, acetylated histone H3 • PH3, phosphohistone H3 • Calu-6, human epithelial-like anaplastic carcinoma (probably lung) • Colo 205, SW 620 and LoVo, epithelial-like human colon adenocarcinoma • EACC, European Collection of Cell Cultures • ATCC, American Type Culture Collection • PD, pharmacodynamic • H&E, hematoxylin and eosin • DAB, diaminobenzadine • IMS, industrial methylated spirits • HLS, hue, luminosity, saturation
Histones are important regulators of gene activity through integration into gene transcriptional machinery and dynamic posttranscriptional modification. It is now well known that levels of histone acetylation, at a given region of chromatin, correlate with transcriptional activity (Grunstein, 1997). Histone acetyltransferases (HATs) interact with histones or with specific transcription factors to enable transcriptional activation of key cell cycle-associated genes involved in tumor-related pathways; such as cyclin-dependent kinase inhibitor p21WAF/CIP1, or p53 and GATA-1 (Yang and Seto, 2003; Vigushin and Combes, 2002). Histone deacetylase inhibitors (HDIs) mimic the action of HATS in that their activity leads to chromatin open structure and further gene transcription. Interestingly, micro-array profiling has shown that HDIs modulate only a small subset of genes (2–5%). However, most of these genes are involved in several key pathways closely linked to cell cycle, and furthermore, can function aberrantly in cancer (Marks et al., 2004). Thus, HDIs, which antagonise histone deacetylases (HDACs) and induce histone acetylation, appear to have a huge potential for cancer therapy through disruption of cell cycle regulatory and survival-related pathways. In certain cancers, such as leukemias, HDIs also appear to act specifically on tumor cells, yet are relatively non toxic to normal cells (Insinga et al., 2005). Structurally diverse HDIs have shown anti-tumor effects in vitro, in in vivo xenograft models and in clinical trials (Jaboin et al., 2002; Marks et al., 2004). Although their precise mode of action is not fully understood, it appears to involve blockage of one or several apoptotic and cell cycle modifying pathways dependent on dose administered, time of treatment, or tumor type (Marks et al., 2004). The purpose of this pilot study was to use immunohistochemistry to evaluate the pharmacodynamic (PD) effects of "Compound 1," a HDI, in different tumor xenografts (Calu-6, Colo 205, SW 620, and LoVo). This was to form a basis for subsequent suitable immunohistochemical analyses over time in larger numbers of xenografts. Based on the known actions of HDIs with regard to increasing acetylation and subsequent downstream effects on cell cycle regulatory mechanisms, we hypothesized that this inhibitor could potentially act in tumor xenografts to increase acetylation, decrease proliferation, arrest cell cycle and induce apoptosis. These processes were quantitatively measured at different time points following compound administration. The markers used to follow these cellular processes were antibodies against acetylated histone H3 (AcH3), Ki-67 (proliferation), phosphohistone H3 (PH3) (mitosis), and cleaved caspase-3 (apoptosis).
Xenograft Models Human primary colorectal cell lines of metastatic origin (Colo 205, SW 620, LoVo) and an anaplastic carcinoma probably of lung origin (Calu-6) were injected subcutaneously into nude mice as previously established under U.K. Home Office regulations. The cell sources for Colo 205, SW 620, LoVo and Calu-6 were the European Collection of Cell Cultures (EACC, Health Protection Agency, Porton Down, UK) CCL222, EACC CCL227, EACC CCL229 and American Type Culture Collection (ATCC, Manassas, VA) HTB-56, respectively. There were 2 animals per time point for each xenograft apart from the LoVo xenograft, which was implanted in 3 animals per time point. A small number of animals was used in this study in line with the purpose of the work, which was to develop methodology applicable for screening, by immunohistochemistry, for a PD effect of a potential drug. Due to the labour-intensive nature of the development of methodologies suitable for screening, together with the evaluation of antibodies for their potential use, and ethical issues regarding animal usage, only 2 or 3 animals were used. The xenografts used were standard established quality-controlled in-house models used in early evaluation of PD markers and form a basis for further robust model development with compounds in the later lead optimisation stages of drug development. Tumors grew between 10 and 20 days: Calu-6 (10 days), Calu-6 (21 days), LoVo (11 days), Colo 205 (14 days), and SW 620 (18 days). When the tumors were palpable (reaching an approximate volume of 1 cm3), animals were dosed orally with "Compound 1" (50 mg/kg single dose). This dose was chosen based on doses used in previous tumor efficacy work-up and from current literature. Control animals received vehicle only. At 6, 24, and 48 hours post-dosing, animals were sacrificed (in 5% CO2 chamber) and xenografts removed. A skin sample was also excised from each animal at a site distant from the tumor.
Histological Assessment
Immunohistochemistry
Primary Antibodies
Secondary Antibodies
Staining Procedures Ki-67 Mib1 immunohistochemistry was performed using a Lab Vision autoimmunostainer (Model LV-1, Lab Vision UK Ltd., Newmarket). Sections were deparaffinized through 2 washes of xylene and rehydrated in industrial methylated spirit (I.M.S.) (75%), I.M.S (95%) and deionized water (all spirits purchased from Fisher Scientific, Loughborough, UK). Endogenous peroxidase activity was blocked by 3% hydrogen peroxide (H2O2) (Fisher Scientific) for 10 minutes after the pretreatment and slides were washed out in deionized water (dH2O). Ki-67 Mib-1 clone and relevant IgG1 negative control (Dako Cytomation Mouse IgG1, catalog no. X0931, diluted to the same mouse IgG concentration as the primary antibody) were used on appropriate tissue sections. The sections were re-hydrated in dH2O, 95% I.M.S., 75% I.M.S., and 2 washes of xylene, then coverslip mounted using a CV5000 coverslipper (Leica Microsystems UK Ltd., Milton Keynes).
Qualitative Microscopic Examination
Quantitative Image Analysis
Cleaved Caspase-3
PH3 Mitosis
PH3 Metaphase Cells
Histological Description of Xenografts All xenografts were predominantly in a dermal location and, in general, formed a well-defined spherical to ovoid mass.
Acetylation
Skin Increased acetylation was observed in epidermal keratinocytes, hair follicles and sebaceous glands at 6 hours following compound administration (Figure 3).
Apoptosis (Cleaved Caspase-3) "Compound 1" increased apoptosis in SW 620 xenografts and slightly in LoVo xenografts at 6–24 hours compared with controls (Figure 4). There was no difference in apoptosis between control and treated Colo 205 and Calu-6 xenografts. In addition, in the skin of animals from 1 study at the 6-hour time point, we observed an increased number of apoptotic cells in hair follicles compared to control animals (Figure 5).
PH3 (Mitosis) PH3 is a marker for cells in mitosis. Following drug treatment, a decrease in PH3 expression occurred at 6–48 hours in the SW 620 xenograft only (Figure 6a). In other xenografts there was no difference between control and treated xenografts.
PH3 (Metaphase Assessment) An increase in metaphase figures is an indication of G1 to S cell cycle phase arrest and may result from a backup of cells in metaphase unable to move forward (Figure 6b). A moderate increase in the number of mitotic metaphase figures was observed in the SW 620 xenograft only, between 24–48 hours following treatment (Figure 6c).
The immunohistochemical evaluation of acetylation, apoptosis, mitosis and proliferation in xenografts following a single administration of a histone deacetylase inhibitor, "Compound 1," highlights the benefits of such an approach for determining the mode of action of these compounds in the commonly used nude mouse xenograft model. This initial work was carried out as a pilot PD study using small numbers of animals to confirm acetylation in vivo following early indications that "Compound 1" acted chemically as an HDI. Based on current literature that HDIs may act to alter cell cycle progression, proliferation and apoptosis (Marks et al., 2001), we also examined these cellular functions using IHC. "Compound 1" clearly increases acetylation of histone H3 within all xenografts examined and this effect is consistent with the predicted response. Using both tumor and skin for analysis, HDI effect is demonstrated by an increase in acetylation in xenografts at 6–24 hours post compound administration and also in skin at the 6-hour time point. The use of acetylation as a biomarker of pharmacodynamic activity of HDIs in the clinic has been applied to blood and bone marrow samples of patients with acute promyelocytic leukemia (APL) (Warrell et al., 1998). Our work indicates that tumor biopsy and skin samples might also be used for a similar purpose. Immunohistochemical measurement of proliferation by measuring Ki-67 expression has been widely used as a method for assessment of tumor biological behaviour. Ki-67 antigens are present in proliferating cells (in G1, S, G2, and mitosis cell division phases) while quiescent or resting cells do not express Ki-67 protein. Determination of levels of Ki-67 expression can be of particular importance in cancers in which the clinical course is difficult to predict by histological criteria alone. Ki-67 expressing-cells were present in all control and treated xenograft sections evaluated in this study. A prominent redistribution of Ki-67 protein occurs during mitosis and we observed a nuclear and nucleolar staining pattern, also previously reported by Scholzen and Gerdes (2000). Qualitative assessment of control and treated xenografts did not reveal a clear difference in numbers of positive cells and further image analysis on Ki-67 stained sections was not carried out. The time points that were measured were acute relative to HDI administration, and it is likely that this antibody may be better suited to evaluate proliferation changes over longer duration efficacy studies. Ki-67 is also a difficult marker to interpret with regard to proliferation as it is not necessarily the case that a cell down-regulates Ki-67 just because DNA synthesis may be blocked. In addition, Ki-67 immunohistochemistry is particularly sensitive to the conditions used for fixation and staining and can sometimes prove difficult to interpret (Scholzen and Gerdes, 2000). We thus decided to use the PH3 antibody to evaluate mitosis in xenografts. This antibody only detects cells in mitosis and thus is not a broad indicator of effect on proliferation. However, our results have shown that the PH3 antibody produces specific, reproducible nuclear staining and is thus ideal for image analysis of nuclear immunopositivity. There was a significant decrease in mitotic cells in only one xenograft, SW 620. This was observed at 6–48 hours after "Compound 1" administration. HDIs have been shown to block cell proliferation by up-regulating the expression of the cyclin dependent kinase inhibitor, p21, thus inducing a G1 phase arrest in a range of tumor types (Saito et al., 1999). We thus decided to evaluate the number of metaphase cells as a potential measure of G1/S arrest in the SW 620 xenograft in which a decrease in mitotic cells following HDI administration had been demonstrated (Figure 6a). An increase in mitotic figures was observed at 48 hours posttreatment in the SW 620 xenograft supporting the view that a G1 to S phase arrest is taking place and that cells cannot move forward from metaphase. Although there is evidence that HDIs can trigger a G2 to M phase arrest (Qiu et al., 2000), a cell block at this stage would result in a decrease in metaphase figures and an increase in polyploid cells with larger than normal nuclei. This was not observed in the present study. However, this data requires further support by particular stage-specific analysis of mitotic cells, for example using Feulgen’s technique (Sudbo et al., 2001). Apoptosis is another potential mechanism through which HDIs may act although there are different and often conflicting conclusions as to which apoptotic pathways are evoked. Some studies indicate a death receptor pathway, such as the induction of Fas or Fas ligand, while other studies suggest that the mitochondrial apoptotic pathway is involved (Rosato et al., 2003). Caspase activity has also been indicated as an integral part of HDI-induced apoptosis, while other papers dispute this (Kwon et al., 2002; Insinga et al., 2005). In the current study, there was evidence of increased apoptosis in one xenograft line, SW 620. The fact that one xenograft, SW 620, shows both an increase in G1-arrested cells and apoptotic cells, may indicate that those cells that undergo cell cycle arrest (cytostasis) do not go through mitosis any further, but die in that stage. Interestingly, apoptotic cells in hair follicles were observed in the skin of 1 study following HDI administration, suggesting the possible use of cleaved caspase-3 immunohistochemistry as a potential skin biomarker in the clinic. Hair follicles staining for cleaved caspase-3 have also been noticed with some other HDI compounds (J. McKay, personal communication, June 2004).
Our pilot study has given an insight into the mode of action of one histone deacetylase inhibitor on human xenografts implanted into nude mice. Immunohistochemistry was used to measure acetylation, proliferation, mitosis and apoptosis 6–48 hours following a single dose of one compound, to investigate possible mode of action and provide background information for further tumor PD and efficacy studies with similar compounds in a more advanced stage of development. These particular cellular processes were chosen based on current published information available on in vitro and clinical activity of HDIs in development for cancer therapy. In addition, image analysis of these processes provided a quantifiable basis of measuring such processes in the future with larger animal numbers. Time-point evaluation following a single dose also provided a model to dissect out direct effects on particular aspects of cell cycle and progression without the complications of longer-term compound administration that could mask these early responses. Acetylation has been reproducibly increased in all xenografts examined. However, the variable effect on proliferation, mitosis, apoptosis, and cell cycle arrest in different xenografts highlights how the downstream actions of the HDIs are highly dependent on cell type and time of administration of HDI. Our work has highlighted that in the evaluation of these types of compounds in efficacy studies, consideration must be given to the fact that although their anti-tumor effects appear to involve common mechanisms, particularly disruption of cell cycle events and disruption of cytoprotective signalling pathways or mitochondrial injury and apoptosis, the actions are multiple and can be variable. Other histone modifications are becoming apparent such as phosphorylation, methylation and ubiquitination (Kristeleit et al., 2004) and these, in time, may also have to be evaluated in our mode of action studies. This study has also highlighted the use of immunohistochemistry to detect acetylation and apoptosis in skin following HDI administration, and is clearly of relevance in the development of other PD markers to monitor in the clinic.
Thanks to A. Bigley and her IHC team for help with method development for imaging studies, C. Chresta and S. East for help and advice, and D. Godwin for statistical advice. Thanks to Professor J. K. Chipman and Dr. N. Hodges for their encouragement on the MRes Molecular Mechanistic Toxicology course at Birmingham University, United Kingdom. This project was supported by AstraZeneca Safety Assessment Pathology Department, Alderley Park, Cheshire, England, United Kingdom.
Toxicologic Pathology, Vol. 33, No. 7,
792-799 (2005) This article has been cited by other articles:
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Abstract
Introduction
