Cell Cycle-associated Gene Expression Research Articles

Regulatory chromatin rewiring promotes metabolic switching during adaptation to oncogenic receptor tyrosine kinase inhibition

Oesophageal adenocarcinoma (OAC) patients show poor survival rates and there are few targeted molecular therapies available. However, components of the receptor tyrosine kinase (RTK) driven pathways are commonly mutated in OAC, typified by high frequency amplifications of the RTK ERBB2. ERBB2 can be therapeutically targeted, but this has limited clinical benefit due to the acquisition of drug resistance. Here we examined how OAC cells adapt to ERBB2 inhibition as they transition to a drug resistant state. ERBB2 inhibition triggers widespread remodelling of the accessible chromatin landscape and the underlying gene regulatory networks. The transcriptional regulators HNF4A and PPARGC1A play a key role in this network rewiring. Initially, inhibition of cell cycle associated gene expression programmes is observed, with compensatory increases in the programmes driving changes in metabolic activity. Both PPARGC1A and HNF4A are required for the acquisition of resistance to ERBB2 inhibition and PPARGC1A is instrumental in promoting a switch to dependency on oxidative phosphorylation. Our work therefore reveals the molecular pathways that support the acquisition of a resistant state and points to potential new therapeutic strategies to combat cellular adaptation and ensuing drug resistance.

The Phenoxyphenol Compound diTFPP Mediates Exogenous C2-Ceramide Metabolism, Inducing Cell Apoptosis Accompanied by ROS Formation and Autophagy in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is a severe disease that accounts for 80% of liver cancers. Chemotherapy is the primary therapeutic strategy for patients who cannot be treated with surgery or who have late-stage HCC. C2-ceramide is an effective reagent that has been found to inhibit the growth of many cancer types. The metabolism of C2-ceramide plays a vital role in the regulation of cell death/cell survival. The phenoxyphenol compound 4-{2,3,5,6-tetrafluoro-4-[2,3,5,6-tetrafluoro-4-(4-hydroxyphenoxy)phenyl]phenoxy}phenol (diTFPP) was found to have a synergistic effect with C2-ceramide, resulting in considerable cell death in the HA22T HCC cell line. diTFPP/C2-ceramide cotreatment induced a two- to threefold increase in cell death compared to that with C2-ceramide alone and induced pyknosis. Annexin V/7-aminoactinomycin D (7AAD) double staining and Western blotting indicated that apoptosis was involved in diTFPP/C2-ceramide cotreatment-mediated cell death. We next analyzed transcriptome alterations in diTFPP/C2-ceramide-cotreated HA22T cells with next-generation sequencing (NGS). The data indicated that diTFPP treatment disrupted sphingolipid metabolism, inhibited cell cycle-associated gene expression, and induced autophagy and reactive oxygen species (ROS)-responsive changes in gene expression. Additionally, we assessed the activation of autophagy with acridine orange (AO) staining and observed alterations in the expression of the autophagic proteins LC3B-II and Beclin-1, which indicated autophagy activation after diTFPP/C2-ceramide cotreatment. Elevated levels of ROS were also reported in diTFPP/C2-ceramide-treated cells, and the expression of the ROS-associated proteins SOD1, SOD2, and catalase was upregulated after diTFPP/C2-ceramide treatment. This study revealed the potential regulatory mechanism of the novel compound diTFPP in sphingolipid metabolism by showing that it disrupts ceramide metabolism and apoptotic sphingolipid accumulation.

Molecular profiling reveals a hypoxia signature in breast implant-associated anaplastic large cell lymphoma

Breast implant-associated anaplastic large cell lymphoma (BIAALCL) is a recently characterized T-cell malignancy that has raised significant patient safety concerns and led to worldwide impact on the implants used and clinical management of patients undergoing reconstructive or cosmetic breast surgery. Molecular signatures distinguishing BIA-ALCL from other anaplastic large cell lymphomas have not been fully elucidated and classification of BIA-ALCL as a World Health Organization entity remains provisional. We performed RNA sequencing and gene set enrichment analysis comparing BIA-ALCL to non-BIAALCL and identified dramatic upregulation of hypoxia signaling genes including the hypoxia-associated biomarker CA9 (carbonic anyhydrase- 9). Immunohistochemistry validated CA9 expression in all BIA-ALCL, with only minimal expression in non-BIA-ALCL. Growth induction in BIA-ALCL-derived cell lines cultured under hypoxic conditions was proportional to upregulation of CA9 expression, and RNA sequencing demonstrated induction of the same gene signature observed in BIAALCL tissue samples compared to non-BIA-ALCL. CA9 silencing blocked hypoxia-induced BIA-ALCL cell growth and cell cycle-associated gene expression, whereas CA9 overexpression in BIA-ALCL cells promoted growth in a xenograft mouse model. Furthermore, CA9 was secreted into BIA-ALCL cell line supernatants and was markedly elevated in human BIA-ALCL seroma samples. Finally, serum CA9 concentrations in mice bearing BIA-ALCL xenografts were significantly elevated compared to those in control serum. Together, these findings characterize BIA-ALCL as a hypoxia-associated neoplasm, likely attributable to the unique microenvironment in which it arises. These data support classification of BIA-ALCL as a distinct entity and uncover opportunities for investigating hypoxia-related proteins such as CA9 as novel biomarkers and therapeutic targets in this disease.

TNF-α Differentially Regulates Cell Cycle Genes in Promyelocytic and Granulocytic HL-60/S4 Cells.

Tumor necrosis factor alpha (TNF-α) is a potent cytokine involved in systemic inflammation and immune modulation. Signaling responses that involve TNF-α are context dependent and capable of stimulating pathways promoting both cell death and survival. TNF-α treatment has been investigated as part of a combined therapy for acute myeloid leukemia due to its modifying effects on all-trans retinoic acid (ATRA) mediated differentiation into granulocytes. To investigate the interaction between cellular differentiation and TNF-α, we performed RNA-sequencing on two forms of the human HL-60/S4 promyelocytic leukemia cell line treated with TNF-α. The ATRA-differentiated granulocytic form of HL-60/S4 cells had an enhanced transcriptional response to TNF-α treatment compared to the undifferentiated promyelocytes. The observed TNF-α responses included differential expression of cell cycle gene sets, which were generally upregulated in TNF-α treated promyelocytes, and downregulated in TNF-α treated granulocytes. This is consistent with TNF-α induced cell cycle repression in granulocytes and cell cycle progression in promyelocytes. Moreover, we found evidence that TNF-α treatment of granulocytes shifts the transcriptome toward that of a macrophage. We conclude that TNF-α treatment promotes a divergent transcriptional program in promyelocytes and granulocytes. TNF-α promotes cell cycle associated gene expression in promyelocytes. In contrast, TNF-α stimulated granulocytes have reduced cell cycle gene expression, and a macrophage-like transcriptional program.

Abstract P3-10-08: Markers of response to CDK4 & 6 inhibition from neoMONARCH: A phase II neoadjuvant study of abemaciclib in postmenopausal women with hormone receptor positive, HER2 negative breast cancer

Abstract Background: Combination treatments of endocrine therapy (ET) with CDK4 & 6 inhibitors have improved outcomes in patients with HR+ advanced breast cancer, both as initial therapy and after progression on ET. Abemaciclib is a selective inhibitor of CDK4 & 6 approved on a continuous dosing schedule for the treatment of HR+, HER2- MBC patients (pts), alone or in combination with ET. However, biomarkers that predict benefit from this class of agents remain elusive. We previously reported in the phase II neoadjuvant neoMONARCH study (NCT02441946), after 2 weeks of treatment, abemaciclib, alone or in combination with anastrozole (ANZ), led to a significantly higher rate of complete cell cycle arrest (CCCA, defined as Ki67 ≤2.7%) compared to ANZ alone in early stage HR+, HER2- breast cancer (Martin et al. SABCS 2017). As an exploratory aim of this trial, we evaluated the gene expression analyses in order to determine markers of sensitivity and resistance to therapy. Methods: Serial biopsies were collected at 3 time points: Baseline (BL) - prior to treatment, Early – after 2 weeks of therapy with abemaciclib, ANZ, or abemaciclib+ANZ, and Late – after 2 weeks of initial therapy followed by 14 weeks of abemaciclib+ANZ. RNA was extracted from FFPE tumor biopsies at each timepoint and subjected to a Cell Cycle Associated Gene (CCAG) expression panel using the Modaplex® platform and whole transcriptome RNA sequencing. Ki67 was measured at each time point by immunohistochemistry (IHC). Tumors were categorized by the post-treatment Ki67 expression as either sensitive (Ki67 ≤2.7) or resistant (Ki67 ≥7.4), based upon the IMPACT and POETIC studies. Additionally, tumors intrinsically resistant/sensitive to therapy were also identified. Results: ANZ-treated tumors that did not achieve CCCA at 2 weeks (N= 8) displayed higher expression of the cell cycle associated genes FOXM1, E2F1, TOPO2A, and RRM2. The addition of abemaciclib to ANZ decreased gene expression in a majority of the tumors (N=5, 62.5%). Tumors intrinsically resistant to treatment with abemaciclib+ANZ displayed persistently elevated levels of cell cycle associated genes compared to sensitive tumors. Finally, gene expression signature of Rb loss-of-function (Rbsig) and RB1 gene expression levels were associated with sensitivity to abemaciclib. Conclusion: On-treatment Ki67 indicated treatment sensitivity and correlated with cell cycle associated gene expression in sensitive and resistant tumors. These exploratory analyses suggest that gene expression analyses may identify genomic markers for abemaciclib and ET treatment sensitivity and may help inform in which tumors to use abemaciclib. Citation Format: Hurvitz S, Martin M, Wijayawardana S, Brahmachary M, Ebert PJ, Young S, Jansen V, Slamon D. Markers of response to CDK4 & 6 inhibition from neoMONARCH: A phase II neoadjuvant study of abemaciclib in postmenopausal women with hormone receptor positive, HER2 negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-08.

Abstract 343: Targeting DNA replication as a therapeutic strategy for acute myeloid leukemia with DNMT3A mutations

Abstract Acute Myeloid Leukemia (AML) is an aggressive malignancy of the blood system, wherein somatic mutations in epigenetic modifier and chromatin remodeling genes are common. Recurrent somatic mutations in the DNA methyltransferase 3 alpha (DNMT3A) gene are detected in about 30% of AML cases, and predict poor outcomes in patients treated with anthracycline-based induction chemotherapy, due to suboptimal response to therapy and subsequent early disease relapse. DNA methylation profiling in both primary AML samples and in animal models carrying a DNMT3A mutation detected modest DNA hypomethylation that did not fully explain how mutant DNMT3A contributed to AML pathogenesis. Recent studies have uncovered disordered nucleosome remodeling in response to DNA topological stress in DNMT3A-mutant cells, which was most pronounced during DNA replication. These changes were accompanied by negative enrichment of the cell cycle-associated gene expression signatures and attenuated CHK1 signaling, implicated in DNA damage response and replication fork integrity. Therefore, we hypothesized that targeting the S-phase in leukemic cells with DNMT3A mutations by pharmacologic agents that stall replication may offer enhanced therapeutic benefit. Indeed, our studies show increased sensitivity to replication stalling pharmacological treatments such as nucleoside analogues cytarabine and fludarabine in different cellular systems harboring DNMT3A mutations. This was accompanied by increased apoptosis as measured by PARP cleavage and annexin V binding.Analysis of the DNA damage signaling revealed elevated CHK1 phosphorylation signaling and accumulation of the DNA damage marker gammaH2A.X. A similar drug sensitivity trend was observed in murine Dnmt3a-mutant bone marrow cells treated with cytarabine, using clonogenic survival in MethoCult as readout. In order to examine this, we are developing isogenic cellular systems with differing DNMT3A mutational status via the CRISPR-Cas9 gene-editing approach. Further, these isogenic cell lines will be used as a tool to characterize the effect of mutant DNMT3A on replication progression, arrest, and resolution under pharmacologically induced replication stress. This will include analysis of the chromatin remodeling at the replication forks, proficiency of DNA damage repair, and dynamic localization of wild-type and mutant DNMT3A within the cells throughout the cell cycle. Finally, we will test the efficacy of replication stress inducing therapies in vivo in a genetically accurate mouse model of Dnmt3a-mutant AML. These studies will shed light on the mechanism of a potential vulnerability in DNMT3A-mutant leukemia cells and may lead to a more effective treatment strategy for this common subtype of AML. Citation Format: Kartika Venugopal, Daphné Dupéré-Richer, Jonathan Licht, Olga Guryanova. Targeting DNA replication as a therapeutic strategy for acute myeloid leukemia with DNMT3A mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 343.

Unravelling Cell Cycle and Ontogeny Transcriptional Heterogeneity in Hematopoietic Stem Cells through Integrated Single Cell RNA-Seq

During fetal development, hematopoietic stem cells (HSCs) undergo a remarkable expansion through a combination of rapid proliferation and high rates of self-renewal. In contrast, adult HSCs are characterized by long-term quiescence. Understanding of the molecular mechanisms underlying these ontogeny-dependent differences in cell cycle and self-renewal is hampered by marked heterogeneity within the HSC compartment, making it difficult to distinguish overlapping signatures in bulk transcriptional data. Advances in single cell genomics provide a new opportunity to tease apart different sources of gene expression heterogeneity, including those relating to cell cycle and self-renewal capability.To address these questions, and improve resolution for cell-cycle annotation of individual HSCs, we developed an integrated single cell (sc)RNA-seq and live cell-cycle staining technique using Hoechst 33342 (DNA) and Pyronin-y (RNA) based FACS index sorting, followed by smart-seq2 based scRNA-seq. We validated our approach on 4 hematopoietic cell lines from mouse and human, using these data as a training set to apply a novel integrated pseudotime package that orders single cells by stage of cell cycle rather than developmental trajectory. By this approach we detected non-canonical cell cycle genes not apparent through bulk sorting of distinct cell cycle phases, and not previously annotated in published cell cycle gene sets (sc-pseudotime genes = 665, FDR < 0.05; non-annotated cell cycle = 487; non-bulk detected = 570).We then applied our technique to analyze primary mouse HSCs from 3 developmental time points (e15.5 fetal liver (FL), 2 week old bone marrow, and 6 week old adult bone marrow (ABM), n >1,500 single cells). Our cell cycle based integrated pseudotime analysis revealed distinct cell cycle signatures for FL, ABM, and common cell cycle related transcripts across distinct developmental time points that have not previously been described, including 555 unique cell cycle genes in FL; 401 unique cell cycle genes in ABM, and 93 novel cell cycle genes in common to both developmental time points e.g. Pclaf, Zfp367 and including long non-coding RNAs e.g. Lockd (FDR < 0.001).Our dataset uniquely allowed us to explore ontogeny related molecular signatures without the overriding effect of confounding cell cycle associated gene expression by directly comparing non-mitotic cells from FL and ABM groups. We identified 404 differentially expressed transcripts (FDR <0.001, >2FC), including genes of unknown HSC function (e.g., FL: Lgals1, Gmfg; ABM: Zfp36l1, Rgs1). Single cell qPCR confirmed aberrant expression of 26/29 (89.7%) selected ontogeny candidate genes. Furthermore, hallmark gene set enrichment analysis revealed upregulation of oxidative phosphorylation, MYC targets, and E2F targets in FL; and TNFA signaling, Hypoxia, and TGF-beta signaling, among others, in ABM (FDR <0.01, NES > 1.5).We then functionally reversed ABM quiescence through in vivo 5-FU treatment, and performed our single cell RNA-seq analysis on HSCs both 2 and 6 days post injection. This allowed us to identify genes and pathways associated with selective resistance of HSCs to chemotherapy, including upregulation of the hallmark gene sets for unfolded protein response, fatty acid metabolism, and MTOR signaling (FDR < 0.01, NES > 1.5 for each set).To functionally validate novel ontogeny related genes we utilized genetic mouse models for two unexplored ABM related genes, Zfp36L1, an RNA-binding zinc finger protein, and Rgs1 a regulator of G-protein coupled signaling. We transplanted Cre-ERT2 conditionally floxed Zfp36L1 bone marrow with CD45.1 competitor control and induced deletion by tamoxifen four weeks post transplant. Compared to the initial four-week post transplant time point we observed a significant reduction in chimerism from Zfp36L1 deleted bone marrow compared to Cre-ERT2 control (p < .05). Competitive transplantation of Rgs1 -/- and WT bone marrow at a 1:1 ratio with CD45.1 competitors resulted in significantly reduced myeloid chimerism at 16 weeks post transplant. Secondary transplant and single cell cycle molecular analysis of these mice are ongoing together with functional validation of a number of other candidate genes.Our results demonstrate the utility of single cell analysis to discover novel HSC regulators providing a unique dataset for further studies investigating regulators of HSC function. [Display omitted] DisclosuresMead:BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.

PM2.5-induced alterations of cell cycle associated gene expression in lung cancer cells and rat lung tissues.

The aim of the current study was to investigate the expression of cell cycle-associated genes induced by fine particulate matter (PM2.5) in lung cancer cell line and tissues. The pulmonary lymph node metastasis cells (H292) were treated with PM2.5in vitro. Wistar rats were used to perform an in vivo study. Rats were randomly assigned to experiment and control groups and those in the experiment group were exposed to PM2.5 once every 15 d, while those in the control group were exposed to normal saline. The cell cycle-associated genes expression was analyzed by real-time PCR. Trachea and lung tissues of rats were processed for scanning electron microscopic (SEM) examinations. Exposure of H292 cells to PM2.5 dramatically increased the expressions of p53 and cyclin-dependent kinase 2 (CDK2) after 24h of exposure (p<0.01) and markedly increased the expressions of the cell division cycle 2 (Cdc2) and cyclin B after 48h of exposure (p<0.01), while those genes expressions were significantly reduced after 72h of exposure, at which time the expression of p21 was predominant (p<0.01). In vivo studies further demonstrated these results. The results of SEM suggested that both of the trachea and lung tissues were damaged and the degree of damage was time-dependent. In conclusion, PM2.5 can induce significantly alterations of p53 and CDK2 in the early phase, Cdc2 and cyclin B in mid-term and p21 in long-term exposure. The degree of PM2.5-induced damage to the trachea and lung tissue was time-dependent.

Meta-analysis reveals conserved cell cycle transcriptional network across multiple human cell types

BackgroundCell division is central to the physiology and pathology of all eukaryotic organisms. The molecular machinery underpinning the cell cycle has been studied extensively in a number of species and core aspects of it have been found to be highly conserved. Similarly, the transcriptional changes associated with this pathway have been studied in different organisms and different cell types. In each case hundreds of genes have been reported to be regulated, however there seems to be little consensus in the genes identified across different studies. In a recent comparison of transcriptomic studies of the cell cycle in different human cell types, only 96 cell cycle genes were reported to be the same across all studies examined.ResultsHere we perform a systematic re-examination of published human cell cycle expression data by using a network-based approach to identify groups of genes with a similar expression profile and therefore function. Two clusters in particular, containing 298 transcripts, showed patterns of expression consistent with cell cycle occurrence across the four human cell types assessed.ConclusionsOur analysis shows that there is a far greater conservation of cell cycle-associated gene expression across human cell types than reported previously, which can be separated into two distinct transcriptional networks associated with the G1/S-S and G2-M phases of the cell cycle. This work also highlights the benefits of performing a re-analysis on combined datasets.

Silencing miR-16 Expression Promotes Angiotensin II Stimulated Vascular Smooth Muscle Cell Growth.

miRNAs are a class of non-coding endogenous small RNAs that control gene expression at the posttranscriptional level and involved in cell proliferation, migration and differentiation. Dysregulation of miRNA expression is involved in a variety of human diseases including cardiovascular diseases. miRNAs have been shown to regulate vascular smooth muscle cell (VSMC) function and play vital roles in hypertension, restenosis and atherosclerosis. Here we reported that miR-16 as one of miRNAs in the miR-15 family was highly expressed in vascular smooth muscle cells (VSMCs) and involved in angiotensin II (Ang II) mediated VSMC signaling pathways. Ang II downregulated miR-16 expression in VSMCs. Lentiviral vector mediated miR-16 knockdown promoted Ang II-induced cell proliferation and migration. Moreover, silencing miR-16 enhanced Ang II induced cell cycle associated gene expression and promoted Ang II-activated cell proliferative pathways ERK1/2 and p38. Our finding demonstrated for the first time that miR-16 was a potential therapeutic target by participating in the Ang II-associated multiple signaling pathways in cardiovascular diseases.

Nupr1-Aurora Kinase A Pathway Provides Protection against Metabolic Stress-Mediated Autophagic-Associated Cell Death

The limited supply of oxygen and nutrients is thought to result in rigorous selection of cells that will eventually form the tumor. Nupr1 expression pattern was analyzed in human tissue microarray (TMA) and correlated with survival time of the patient. Microarray analysis was conducted on MiaPaCa2 cells subjected to metabolic stress in Nupr1-silenced conditions. DNA repair and cell cycle-associated gene expression was confirmed by real-time quantitative PCR (qRT-PCR). Nupr1 and AURKA protective role were analyzed using RNA interference (RNAi) silencing or overexpression. DNA damage and autophagy were analyzed by Western blot analysis and immunofluorescence. We showed that both Nupr1 and HIF1α are coexpressed in human pancreatic ductal adenocarcinoma (PDAC) samples and negatively correlate with survival time. PDAC-derived cells submitted to hypoxia and/or glucose starvation induce DNA damage-dependent cell death concomitantly to the overexpression of stress protein Nupr1. Affymetrix-based transcriptoma analysis reveals that Nupr1 knockdown enhances DNA damage and alters the expression of several genes involved in DNA repair and cell-cycle progression. Expression of some of these genes is common to hypoxia and glucose starvation, such as Aurka gene, suggesting that Nupr1 overexpression counteracts the transcriptional changes occurring under metabolic stress. The molecular mechanism by which hypoxia and glucose starvation induce cell death involves autophagy-associated, but not caspase-dependent, cell death. Finally, we have found that AURKA expression is partially regulated by Nupr1 and plays a major role in this response. Our data reveal that Nupr1 is involved in a defense mechanism that promotes pancreatic cancer cell survival when exposed to metabolic stress.

AP-2γ promotes proliferation in breast tumour cells by direct repression of the CDKN1A gene

Overexpression of the activator protein (AP)-2gamma transcription factor in breast tumours has been identified as an independent predictor of poor outcome and failure of hormone therapy. To understand further the function of AP-2gamma in breast carcinoma, we have used an RNA interference and gene expression profiling strategy with the MCF-7 cell line as a model. Gene expression changes between control and silenced cells implicate AP-2gamma in the control of cell cycle progression and developmental signalling. A function for AP-2gamma in cell cycle control was verified using flow cytometry: AP-2gamma silencing led to a partial G1/S arrest and induction of the cyclin-dependent kinase inhibitor, p21cip/CDKN1A. Reporter and chromatin immunoprecipitation assays demonstrated a direct, functional interaction by AP-2gamma at the CDKN1A proximal promoter. AP-2gamma silencing coincided with acquisition of an active chromatin conformation at the CDKN1A locus and increased gene expression. These data provide a mechanism whereby AP-2gamma overexpression can promote breast epithelial proliferation and, coupled with previously published data, suggest how loss of oestrogen regulation of AP-2gamma may contribute to the failure of hormone therapy in patients.

Gene Expression Reveals Two Distinct Biological Groups within T-Cell Prolymphocytic Leukaemia.

T-cell prolymphocytic leukemia (T-PLL) is rare and presents with widespread disease. Indolent presentations are seen but eventually progress. The disease shows marked chemoresistance and is best treated with the monoclonal anti-CD52 antibody (CAMPATH). Prolymphocytes show a post-thymic phenotype and are CD4+CD8− (65%), CD4−CD8+ (10%) or CD4+CD8+ (25%). This double positive phenotype, raises questions about the putative ontology of T-PLL. Morphological heterogeneity, with typical (75%), small cell (20%) and cerebriform/sezary-like variants (5%) is described. Inversions or reciprocal translocations of chromosome 14 involving breakpoints at q11 (TCR a/d) and q32.1 (TCL1 and TCL1b) are seen (~ 80%). Other common abnormalities involve chromosome 8, translocation (X;14)(q28;q11) and, ATM (11q23). We investigated the clinico-pathological heterogeneity in T-PLL, at the level of the transcriptome and evaluated the ability of gene expression profiling to sub-classify T-PLL. Total RNA was extracted from blood prolymphocytes (>92% purity) of 22 patients. cDNA synthesis followed by biotin-labelled cRNA synthesis was carried out as per Affymetrix protocols. Fragmented cRNA was hybridized to the Human U133 PLUS2 GeneChip array (54K probes). Microarray services were provided by MRC geneservice (UK HGMP Resource Centre). Hierarchical clustering of samples was performed using a filtered gene set (12,456) and >4 different algorithims. Prediction analysis for micoarray (PAM) and significance analysis of microarray (SAM) were used to evaluate class performance, and partition genes using pre-defined labels of immunophenotype, karyotype, response and morphology. Validation was performed by RT-PCR in a subset of genes.Unsupervised analysis robustly and reproducibly partitioned samples into 2 groups; A (n=8) and B (n=14). SAM analysis identified 4487 differentially expressed transcripts (false discovery rates <1%), >40% of which showed >2-fold difference in expression between the groups. There was no statistical difference in age, immunophenotype or karyotype betweeen groups, however, differential response to CAMPATH was seen. PAM analysis refined a sub-group of ~123 genes which most efficiently differentiated these groups. Group A showed significantly higher rates of non-response and progressive disease as compared to group B (n=14, p=0.036). Key differences related to apoptosis and cell-cycle associated gene expression. Down regulation of caspases (CASP1, CASP2,CASP4, CARD8 and CASP8AP2), cyclins (CCNC, CCND2, CCND3, CCNG1, CCNI, CCNT2), bcl-2, HDAC1, HIPK2, IL6R and ATM were frequent in group A with upregulation of genes implicated in NF-kB (TRAF4, SQSTM1) and TNF pathways (LMNA, ARTS-1), as well as transcription factors such as ATF-3. CD52 expression was ~2-fold higher in group B and may explain in part, differential responses to CAMPATH. RT-PCR validated gene expression data for LMNA and ATF-3. Despite the small numbers, algorithim-independent segregation into 2 consistent groups, in conjunction with the magnitude of gene differences, presence of many mutually exclusive divisions, and low prediciton errors, imply that the 2 identified profiles arise from fundamental differences at a regulatory level and thus likely represent a generalisable classification for T-PLL. Differential responses to CAMPATH may be a sub-feature of this grouping.

Changes in cell cycle-associated gene expression in a model of impaired liver regeneration

Following partial hepatectomy (PH) there is compensatory regeneration of the remnant liver which eventually restores hepatic mass and function. The response to PH was studied in normal BALB/c and athymic nude mice, a model of impaired liver regeneration. Following PH, nude mice demonstrated diminished peak hepatic [ 3H]thymidine uptake and delayed liver mass restoration through 60 h post-PH. However, between 72–120 h there was no significant difference in mass restoration between the groups. The expression of genes associated with different stages of the cell cycle was evaluated in both models. In nude mice, there was an increase in peak expression of c- jun transcripts, while c- myc transcript expression was moderately attenuated. Thymidine kinase (TK) and cyclin-dependent kinase 1 (CDK1) mRNA expression was also diminished in athymic nude mice. The results suggest that while the defect in the regenerative response of the nude mouse after PH affects events in several phases of the cell cycle, mass restoration of the liver is only delayed and not attenuated.