Multiomics approach towards characterization of tumor cell plasticity and its significance in precision and personalized medicine.

Cellular plasticity refers to the ability of cells to change their identity or behavior, which can be advantageous in some cases (e.g., tissue regeneration) but detrimental in others (e.g., cancer metastasis). With a better understanding of cellular plasticity, the complexity of cancer cells, their heterogeneity, and their role in metastasis is being unraveled. The plasticity of the cells could also prove as a nemesis to their characterization. In this review, we have attempted to highlight the possibilities and benefits of using multiomics approach in characterizing the plastic nature of cancer cells. There is a need to integrate fragmented evidence at different levels of cellular organization (DNA, RNA, protein, metabolite, epigenetics, etc.) to facilitate the characterization of different forms of plasticity and cell types. We have discussed the role of cellular plasticity in generating intra-tumor heterogeneity. Different omics level evidence is being provided to highlight the variety of molecular determinants discovered using different techniques. Attempts have been made to integrate some of this information to provide a quantitative assessment and scoring of the plastic nature of the cells. However, there is a huge gap in our understanding of mechanisms that lead to the observed heterogeneity. Understanding of these mechanism(s) is necessary for finding targets for early detection and effective therapeutic interventions in metastasis. Targeting cellular plasticity is akin to neutralizing a moving target. Along with the advancements in precision and personalized medicine, these efforts may translate into better clinical outcomes for cancer patients, especially in metastatic stages.

The role of TAOK3 in cancer progression and development as a prognostic marker: A pan-cancer analysis study.

The protein kinase TAOK3, belongs to the MAP kinase family, is one of three closely related members, namely TAOK1, TAOK2, and TAOK3. We performed a pan-cancer investigation of TAOK3 across different cancer types, including uterine carcinosarcoma, adenocarcinoma of the stomach and pancreas, and endometrial carcinoma of the uterus, to better understand TAOK3's role in cancer. In at least 16 types of cancer, our findings indicate that TAOK3 expression levels differ considerably between normal and tumor tissues. In addition, our study is the first to identify the oncogenic role of TAOK3 locus S331 and S471 in renal clear cell carcinoma, Glioblastoma Multiforme, hepatocellular carcinoma, Lung adenocarcinoma, and Pancreatic adenocarcinoma, indicating their involvement in cancer progression. In addition, our data analysis indicates that copy number variation is the most prevalent form of mutation in the TAOK3 gene, and that there is a negative correlation between TAOK3 mRNA and DNA promoter methylation. Moreover, our analysis suggests that TAOK3 may serve as a prognostic marker for several kinds of cancer, including Colon adenocarcinoma, renal clear cell carcinoma, Lower Grade Glioma, Lung adenocarcinoma, Mesothelioma, and hepatocellular carcinoma. In addition, our research on signature cancer genes has uncovered a positive association between TAOK3 and SMAD2, SMAD4, and RNF168 in most of the malignancies we have examined. TAOK3 is also correlated with the frequency of mutations and microsatellite instability in four types of cancer. Numerous immune-related genes are closely associated with TAOK3 levels in numerous malignancies. TAOK3 expression is positively correlated with immune infiltrates, which include activated CD4 T cells, CD8 T cells, and type 2T helper cells. Our pan-cancer analysis of TAOK3 provides vital insight into its potential role across a variety of cancer types.

Differential Gene Expression Signatures and Cellular Signaling Pathways induced by Lamin A/C Transcript Variants in MCF7 Cell Line.

BACKGROUND: Lamins are the major component of nuclear lamina. Alternative splicing of the 12 exons comprising lamin A/C gene creates five known transcript variants, lamin A, lamin C, lamin AΔ10, lamin AΔ50, and lamin C2. The main objective for this study was to examine the association of critical pathways, networks, molecular and cellular functions regulated by each Lamin A/C transcript variants. METHODS: Ion AmpliSeq Transcriptome Human Gene Expression analysis was performed on MCF7 cells stably transfected with lamin A/C transcript variants. RESULTS: Lamin A or lamin AΔ50 upregulation was associated with activation of cell death and inactivation of carcinogenesis while both lamin C or lamin AΔ10 upregulation activated carcinogenesis and cell death. CONCLUSIONS: Data suggest anti-apoptotic and anti-senescence effects of lamin C and lamin AΔ10 as several functions, including apoptosis and necrosis functions are inactivated following lamin C or lamin AΔ10 upregulation. However, lamin AΔ10 upregulation is associated with a more carcinogenic and aggressive tumor phenotype. Lamin A or lamin AΔ50 upregulation is associated with a predicted activation of increased cell death and inactivation of carcinogenesis. Thus, different signaling pathways, networks, molecular and cellular functions are activated/inactivated by lamin A/C transcript variants resulting in a large number of laminopathies.

Alternatively Spliced Isoforms of MUC4 and ADAM12 as Biomarkers for Colorectal Cancer Metastasis.

There is a pertinent need to develop prognostic biomarkers for practicing predictive, preventive and personalized medicine (PPPM) in colorectal cancer metastasis. The analysis of isoform expression data governed by alternative splicing provides a high-resolution picture of mRNAs in a defined condition. This information would not be available by studying gene expression changes alone. Hence, we utilized our prior data from an exon microarray and found ADAM12 and MUC4 to be strong biomarker candidates based on their alternative splicing scores and pattern. In this study, we characterized their isoform expression in a cell line model of metastatic colorectal cancer (SW480 & SW620). These two genes were found to be good prognostic indicators in two cohorts from The Cancer Genome Atlas database. We studied their exon structure using sequence information in the NCBI and ENSEMBL genome databases to amplify and validate six isoforms each for the ADAM12 and MUC4 genes. The differential expression of these isoforms was observed between normal, primary and metastatic colorectal cancer cell lines. RNA-Seq analysis further proved the differential expression of the gene isoforms. The isoforms of MUC4 and ADAM12 were found to change expression levels in response to 5-Fluorouracil (5-FU) treatment in a dose-, time- and cell line-dependent manner. Furthermore, we successfully detected the protein isoforms of ADAM12 and MUC4 in cell lysates, reflecting the differential expression at the protein level. The change in the mRNA and protein expression of MUC4 and ADAM12 in primary and metastatic cells and in response to 5-FU qualifies them to be studied as potential biomarkers. This comprehensive study underscores the importance of studying alternatively spliced isoforms and their potential use as prognostic and/or predictive biomarkers in the PPPM approach towards cancer.

Presence of Circulatory Autoantibodies Against ROS-Modified Histone H1 Protein in Lymphoma Patients.

Lymphoma is a chronic inflammatory disease in which the immune system is highly affected. Increased oxidative stress is one of the common conditions of cancer and affects macromolecules. Histone modifications affect the chromatin structure and functions. In this study, histone H1 (His-H1) protein was modified by reactive oxygen species (ROS), and structural and chemical changes were studied. Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) patients were selected, and oxidative stress markers, inflammatory cytokines, and serum autoantibodies were analyzed using biochemical and immunological assays. Furthermore, the formation of antigen-antibody immune complexes was assessed by the Langmuir plot. ROS-modified His-H1 (ROS-His-H1) showed substantial structural perturbation in protein (UV-hyperchromicity and increased intrinsic fluorescence) compared to the native His-H1 protein. A possible explanation for the changes is suggested by the exposure of the aromatic chromophore to the solvent. In-depth structural analysis by circular dichroism (CD) exhibited major changes in α-helix (-21.43%) and turns (+33%), reflecting changes in the secondary structure of histone H1 protein after ROS exposure. ELISA and competitive ELISA findings revealed high recognitions of serum autoantibodies to ROS-His-H1 from NHL, followed by HL subjects. Healthy controls showed negligible binding. Non-modified His-H1 did not show any binding with serum samples from either cohort. High apparent association constants (ACCs) were calculated for ROS-His-H1 using purified IgGs from NHL (1.46 × 10-6 M) compared to HL (1.33 × 10-6 M) patients. Non-modified His-H1 exhibited a hundred times less ACC for NHL (2.38 × 10-8 M) and HL (2.46 × 10-8 M) patients. Thus, ROS modifications of histone H1 cause structural changes and expose cryptic neo-epitopes on the protein against which autoantibodies were generated. These perturbations might affect the histone DNA interaction dynamics and potentially be correlated with gene dysregulation. These subtle molecular changes with an immune imbalance might further aggravate the disease.

Implications of zonal architecture on differential gene expression profiling and altered pathway expressions in mandibular condylar cartilage.

Mandibular condylar cartilage (MCC) is a multi-zonal heterogeneous fibrocartilage containing different types of cells, but the factors/mechanisms governing the phenotypic transition across the zones have not been fully understood. The reliability of molecular studies heavily rely on the procurement of pure cell populations from the heterogeneous tissue. We used a combined laser-capture microdissection and microarray analysis approach which allowed identification of differential zone-specific gene expression profiling and altered pathways in the MCC of 5-week-old rats. The bioinformatics analysis demonstrated that the MCC cells clearly exhibited distinguishable phenotypes from the articular chondrocytes. Additionally, a set of genes has been determined as potential markers to identify each MCC zone individually; Crab1 gene showed the highest enrichment while Clec3a was the most downregulated gene at the superficial layer, which consists of fibrous (FZ) and proliferative zones (PZ). Ingenuity Pathway Analysis revealed numerous altered signaling pathways; Leukocyte extravasation signaling pathway was predicted to be activated at all MCC zones, in particular mature and hypertrophic chondrocytes zones (MZ&HZ), when compared with femoral condylar cartilage (FCC). Whereas Superpathway of Cholesterol Biosynthesis showed predicted activation in both FZ and PZ as compared with deep MCC zones and FCC. Determining novel zone-specific differences of large group of potential genes, upstream regulators and pathways in healthy MCC would improve our understanding of molecular mechanisms on regional (zonal) basis, and provide new insights for future therapeutic strategies.

Identification of Novel Mutations in Colorectal Cancer Patients Using AmpliSeq Comprehensive Cancer Panel.

Biomarker discovery would be an important tool in advancing and utilizing the concept of precision and personalized medicine in the clinic. Discovery of novel variants in local population provides confident targets for developing biomarkers for personalized medicine. We identified the need to generate high-quality sequencing data from local colorectal cancer patients and understand the pattern of occurrence of variants. In this report, we used archived samples from Saudi Arabia and used the AmpliSeq comprehensive cancer panel to identify novel somatic variants. We report a comprehensive analysis of next-generation sequencing results with a coverage of >300X. We identified 466 novel variants which were previously unreported in COSMIC and ICGC databases. We analyzed the genes associated with these variants in terms of their frequency of occurrence, probable pathogenicity, and clinicopathological features. Among pathogenic somatic variants, 174 were identified for the first time in the large intestine. APC, RET, and EGFR genes were most frequently mutated. A higher number of variants were identified in the left colon. Occurrence of variants in ERBB2 was significantly correlated with those of EGFR and ATR genes. Network analyses of the identified genes provide functional perspective of the identified genes and suggest affected pathways and probable biomarker candidates. This report lays the ground work for biomarker discovery and identification of driver gene mutations in local population.

Free fatty acids receptors 2 and 3 control cell proliferation by regulating cellular glucose uptake.

BACKGROUND: Colorectal cancer (CRC) is a worldwide problem, which has been associated with changes in diet and lifestyle pattern. As a result of colonic fermentation of dietary fibres, short chain free fatty acids are generated which activate free fatty acid receptors (FFAR) 2 and 3. FFAR2 and FFAR3 genes are abundantly expressed in colonic epithelium and play an important role in the metabolic homeostasis of colonic epithelial cells. Earlier studies point to the involvement of FFAR2 in colorectal carcinogenesis. AIM: To understand the role of short chain FFARs in CRC. METHODS: Transcriptome analysis console software was used to analyse microarray data from CRC patients and cell lines. We employed short-hairpin RNA mediated down regulation of FFAR2 and FFAR3 genes, which was validated using quantitative real time polymerase chain reaction. Assays for glucose uptake and cyclic adenosine monophosphate (cAMP) generation was done along with immunofluorescence studies to study the effects of FFAR2/FFAR3 knockdown. For measuring cell proliferation, we employed real time electrical impedance-based assay available from xCELLigence. RESULTS: Microarray data analysis of CRC patient samples showed a significant down regulation of FFAR2 gene expression. This prompted us to study the FFAR2 in CRC. Since, FFAR3 shares significant structural and functional homology with FFAR2, we knocked down both these receptors in CRC cell line HCT 116. These modified cell lines exhibited higher proliferation rate and were found to have increased glucose uptake as well as increased level of glucose transporter 1. Since, FFAR2 and FFAR3 signal through G protein subunit (Gαi), knockdown of these receptors was associated with increased cAMP. Inhibition of protein kinase A (PKA) did not alter the growth and proliferation of these cells indicating a mechanism independent of cAMP/PKA pathway. CONCLUSION: Our results suggest role of FFAR2/FFAR3 genes in increased proliferation of colon cancer cells via enhanced glucose uptake and exclude the role of PKA mediated cAMP signalling. Alternate pathways could be involved that would ultimately result in increased cell proliferation as a result of down regulated FFAR2/FFAR3 genes. This study paves the way to understand the mechanism of action of short chain FFARs in CRC.

Colorectal cancer in Saudi Arabia as the proof-of-principle model for implementing strategies of predictive, preventive, and personalized medicine in healthcare.

Colorectal cancer (CRC) is the most commonly diagnosed cancer among Saudi males and ranks third in females with up to 73% of cases diagnosed at late stage. This review provides an analysis of CRC situation in the Kingdom of Saudi Arabia (KSA) from healthcare perspective. A PUBMED (1986-2018) search was done to identify publications focusing on CRC in KSA. Due to reports of increased CRC incidence among young age group (< 50), and given the young population of KSA, the disease may burden the national healthcare system in the next decades. Environmental factors attributed to increasing incidence rates of CRC include red meat consumption, sedentary lifestyle, and increased calorie intake. Despite substantial investment in healthcare, attention to predictive diagnostics and targeted prevention is lacking. There is a need to develop national screening guidelines based on evidence that supports a reduction in incidence and mortality of CRC when screening is implemented. Future approaches are discussed based on multi-level diagnostics, risk assessment, and population screening programs focused on the needs of young populations that among others present the contents of the advanced approach by predictive, preventive, and personalized medicine. Recommendations are provided that could help to develop policies at regional and national levels. Countries with demographics and lifestyle similar to KSA may gain insights from this review to shape their policies and procedures.

Elucidating the Reprograming of Colorectal Cancer Metabolism Using Genome-Scale Metabolic Modeling.

Colorectal cancer is the third most incidental cancer worldwide, and the response rate of current treatment for colorectal cancer is very low. Genome-scale metabolic models (GEMs) are systems biology platforms, and they had been used to assist researchers in understanding the metabolic alterations in different types of cancer. Here, we reconstructed a generic colorectal cancer GEM by merging 374 personalized GEMs from the Human Pathology Atlas and used it as a platform for systematic investigation of the difference between tumor and normal samples. The reconstructed model revealed the metabolic reprogramming in glutathione as well as the arginine and proline metabolism in response to tumor occurrence. In addition, six genes including ODC1, SMS, SRM, RRM2, SMOX, and SAT1 associated with arginine and proline metabolism were found to be key players in this metabolic alteration. We also investigated these genes in independent colorectal cancer patients and cell lines and found that many of these genes showed elevated level in colorectal cancer and exhibited adverse effect in patients. Therefore, these genes could be promising therapeutic targets for treatment of a specific colon cancer patient group.

Emerging paradigms in the treatment of liver metastases in colorectal cancer.

Efforts to combat colorectal cancer have benefited from improved screening and surveillance, which facilitates early detection. The survival rate associated with diagnosis at stage I is approximately 90%. However, progress in improving survival in metastatic colorectal cancer (mCRC) has been minimal. This review focuses on mCRC with special emphasis on the molecular aspects of liver metastases, which is one of the most frequently involved organ site. Better molecular evidence is required to guide the decisions for surgical and other interventions used in the clinical management of mCRC. Results from different treatment modalities have exposed significant gaps in the existing paradigms of the mCRC management. Indeed there is a critical need to better understand molecular events and pathways that lead to colorectal cancer liver metastasis. Such a focused approach may help identify biomarkers and drug targets that can be useful in the clinical applications. With this focus, we provide an account of the molecular pathways involved in the spread of CRC to the liver. Specifically, the molecular changes at the DNA and RNA levels that are associated with liver metastases are discussed. Similarly, we describe relevant microRNAs that are identified as regulators of gene expression and can also serve as biomarkers. Conventionally applied biomarkers are not yet specific and sensitive enough to be relied in routine clinical decision making. Hence search for novel biomarkers is critically needed especially if these can be utilized using liquid biopsies. This review provides a comprehensive analysis of current molecular evidence along with potential future directions that could reshape the diagnostic and management paradigms and thus mitigate the devastating impact of colorectal cancer metastasis to the liver.

Effect of sampling procedure on the quality control metrics of cytoscan HD array for studying cytogenetic aspects of colorectal cancer.

OBJECTIVES: The method of colorectal cancer (CRC) tissue sampling would affect its molecular profile and the downstream processing. In this study, we described the impact of CRC tissue sampling procedures on the quality control (QC) metrics of cytoscan HD array. METHODS: We employed a high-resolution cytoscan HD microarray platform to investigate the chromosomal aberrations that could be associated with CRC. We compared the tissue extraction procedures and their impact on the QC parameters from the cytoscan HD array determined by chromosome analysis software (Suite3.1). Median of absolute values of all pairwise differences (MAPD), waviness-standard deviation (Waviness-SD), and single nucleotide polymorphism QC (SNPQC) were the QC parameters that were analyzed. RESULTS: From 67 patients, we collected 843 colorectal tissues. Of these, 65.7% were obtained through endoscopic procedures, and the rest was after surgical resections. The mean transit time between tissue excision and preservation was 26 ± 15.5 and 74.6 ± 24.8 min, respectively. The tissues extracted from the surgical procedure showed mean MAPD of 0.28 ± 0.06 compared to 0.24 ± 0.06, for endoscopy, P = 0.005, degree of waviness-SD of 0.20 ± 0.1 compared to 0.2 ± 0.1, P = 0.64, and SNPQC of 9.6 ± 4.2 compared to 11.1 ± 4.6, P = 0.23. CONCLUSIONS: This report provides objective results that can help in tissue sampling intended to be used for DNA based molecular studies. Tissue collection protocol should be optimized to support microarray-analysis methods. Tissue extraction from endoscopic procedures had faster transit time and relatively better quality metrics outcome than surgical procedures. However, surgical procedures have less refusal rate, higher tissue quantity, and less negative results for malignancy.

Towards personalized medicine of colorectal cancer.

Efforts in colorectal cancer (CRC) research aim to improve early detection and treatment for metastatic stages which could translate into better prognosis of this disease. One of the major challenges that hinder these efforts is the heterogeneous nature of CRC and involvement of diverse molecular pathways. New large-scale 'omics' technologies are making it possible to generate, analyze and interpret biological data from molecular determinants of CRC. The developments of sophisticated computational analyses would allow information from different omics platforms to be integrated, thus providing new insights into the biology of CRC. Together, these technological advances and an improved mechanistic understanding might allow CRC to be clinically managed at the level of the individual patient. This review provides an account of the current challenges in CRC management and an insight into how new technologies could allow the development of personalized medicine for CRC.

A Model of Exposure to Extreme Environmental Heat Uncovers the Human Transcriptome to Heat Stress.

The molecular mechanisms by which individuals subjected to environmental heat stress either recover or develop heat-related complications are not well understood. We analysed the changes in blood mononuclear gene expression patterns in human volunteers exposed to extreme heat in a sauna (temperature of 75.7 ± 0.86 °C). Our analysis reveals that expression changes occur rapidly with no significant increase in core temperature and continue to amplify one hour after the end of heat stress. The reprogramed transcriptome was predominantly inhibitory, as more than two-thirds of the expressed genes were suppressed. The differentially expressed genes encoded proteins that function in stress-associated pathways; including proteostasis, energy metabolism, cell growth and proliferation, and cell death, and survival. The transcriptome also included mitochondrial dysfunction, altered protein synthesis, and reduced expression of genes -related to immune function. The findings reveal the human transcriptomic response to heat and highlight changes that might underlie the health outcomes observed during heat waves.

TXNIP in Metabolic Regulation: Physiological Role and Therapeutic Outlook.

Background & Objective: Thioredoxin-interacting protein (TXNIP) also known as thioredoxin binding protein-2 is a ubiquitously expressed protein that interacts and negatively regulates expression and function of Thioredoxin (TXN). Over the last few years, TXNIP has attracted considerable attention due to its wide-ranging functions impacting several aspects of energy metabolism. TXNIP acts as an important regulator of glucose and lipid metabolism through pleiotropic actions including regulation of ß-cell function, hepatic glucose production, peripheral glucose uptake, adipogenesis, and substrate utilization. Overexpression of TXNIP in animal models has been shown to induce apoptosis of pancreatic ß-cells, reduce insulin sensitivity in peripheral tissues like skeletal muscle and adipose, and decrease energy expenditure. On the contrary, TXNIP deficient animals are protected from diet induced insulin resistance and type 2 diabetes. SUMMARY: Consequently, targeting TXNIP is thought to offer novel therapeutic opportunity and TXNIP inhibitors have the potential to become a powerful therapeutic tool for the treatment of diabetes mellitus. Here we summarize the current state of our understanding of TXNIP biology, highlight its role in metabolic regulation and raise critical questions that could help future research to exploit TXNIP as a therapeutic target.

Filtered selection coupled with support vector machines generate a functionally relevant prediction model for colorectal cancer.

PURPOSE: There has been considerable interest in using whole-genome expression profiles for the classification of colorectal cancer (CRC). The selection of important features is a crucial step before training a classifier. METHODS: In this study, we built a model that uses support vector machine (SVM) to classify cancer and normal samples using Affymetrix exon microarray data obtained from 90 samples of 48 patients diagnosed with CRC. From the 22,011 genes, we selected the 20, 30, 50, 100, 200, 300, and 500 genes most relevant to CRC using the minimum-redundancy-maximum-relevance (mRMR) technique. With these gene sets, an SVM model was designed using four different kernel types (linear, polynomial, radial basis function [RBF], and sigmoid). RESULTS: The best model, which used 30 genes and RBF kernel, outperformed other combinations; it had an accuracy of 84% for both ten fold and leave-one-out cross validations in discriminating the cancer samples from the normal samples. With this 30 genes set from mRMR, six classifiers were trained using random forest (RF), Bayes net (BN), multilayer perceptron (MLP), naïve Bayes (NB), reduced error pruning tree (REPT), and SVM. Two hybrids, mRMR + SVM and mRMR + BN, were the best models when tested on other datasets, and they achieved a prediction accuracy of 95.27% and 91.99%, respectively, compared to other mRMR hybrid models (mRMR + RF, mRMR + NB, mRMR + REPT, and mRMR + MLP). Ingenuity pathway analysis was used to analyze the functions of the 30 genes selected for this model and their potential association with CRC: CDH3, CEACAM7, CLDN1, IL8, IL6R, MMP1, MMP7, and TGFB1 were predicted to be CRC biomarkers. CONCLUSION: This model could be used to further develop a diagnostic tool for predicting CRC based on gene expression data from patient samples.

The anticancer activity of the substituted pyridone-annelated isoindigo (5'-Cl) involves G0/G1 cell cycle arrest and inactivation of CDKs in the promyelocytic leukemia cell line HL-60.

BACKGROUND/AIMS: The antileukemic potential of isoindigos make them desired candidates for understanding their mechanism of action. We have recently synthesized a novel group of pyridone-annelated isoindigos and identified the derivative 5'-Cl that is cytotoxic to various cancer cell lines. In the present study, we analyzed the effect of this compound on cell cycle of the promyelocytic leukemia cell line HL-60. METHODS: HL-60 cells were treated with 5'-Cl and its effect on cell cycle stages were determined by flow cytometry. Expression of cyclins, cyclin dependent kinases (CDKs) and cyclin kinase inhibitors (CKIs) were determined by Western blotting, and activation of CDKs was studied using kinase assays. RESULTS: 5'-Cl remarkably arrested cell cycle in HL-60 cells at the G0/G1 phase in a dose and time-dependent manner. Furthermore, 5'-Cl treatment significantly inhibited expression of D-cyclins, CDK2 and CDK4 and suppressed phosphorylation of the retinoblastoma protein Rb, whereas it increased the level of CKI p21. Molecular modelling experiments show that 5'-Cl may compete with ATP for binding to the catalytic subunit of CDK2 and CDK4 that could lead to inhibition of these enzymes. Indeed, 5'-Cl inhibited the kinase activity of CDK2 and CDK4 both in cell free systems and in treated cells. 5'-Cl also inhibited cell cycle progression in several other tumor cell lines. CONCLUSION: We demonstrate the potent inhibitory effects of 5'-Cl on HL-60 cells could be mediated by arresting cells in the G0/G1 phase.