Key Cell Cycle Regulatory Genes Research Articles

Transcription Regulation of Cell Cycle Regulatory Genes Mediated by NtrX to Affect Sinorhizobium meliloti Cell Division

The cell division of the alfalfa symbiont, Sinorhizobium meliloti, is dictated by a cell cycle regulatory pathway containing the key transcription factors CtrA, GcrA, and DnaA. In this study, we found that NtrX, one of the regulators of nitrogen metabolism, can directly regulate the expression of ctrA, gcrA, and dnaA from the cell cycle pathway. Three sets of S. meliloti ntrX mutants showed similar cell division defects, such as slow growth, abnormal morphology of some cells, and delayed DNA synthesis. Transcription of ctrA and gcrA was upregulated, whereas the transcription of dnaA and ftsZ1 was downregulated in the insertion mutant and the strain of Sm1021 expressing ntrXD53E. Correspondingly, the inducible transcription of ntrX activates the expression of dnaA and ftsZ1, but represses ctrA and gcrA in the depletion strain. The expression levels of CtrA and GcrA were confirmed by Western blotting. The transcription regulation of these genes requires phosphorylation of the conserved 53rd aspartate in the NtrX protein that binds directly to the promoter regions of ctrA, gcrA, dnaA, and ftsZ1 by recognizing the characteristic sequence CAAN2-5TTG. Our findings suggest that NtrX affects S. meliloti cell division by regulating the transcription of the key cell cycle regulatory genes.

Abstract DP-008: SILICA GEL ENCAPSULATION AS A POTENTIAL TOOL TO IDENTIFY DORMANT AND DRUG-RESISTANT CELL POPULATIONS IN OVARIAN CANCER

Abstract Despite advances in standard of care treatment, a significant number of ovarian cancer patients will exhibit disease recurrence, either due to the presence of chemoresistant cells or the reawakening of dormant tumor cells. Currently there are no methods to predict which patients are likely to recur, and the mechanisms that allow a unique subpopulation of cancer cells to enter dormancy and survive chemotherapy remain unclear. Since chemotherapy is more effective at destroying highly proliferative cells, it is theorized that tumor cells capable of entering a dormant state are also the same population able to evade initial treatment and ultimately engender chemoresistance. A silica gel encapsulation platform that uses physical confinement to inhibit proliferation has previously demonstrated the ability to distinguish between dormancy-capable and dormancy-incapable breast cancer cell lines. In this work, we sought to apply this platform to an ovarian cancer cell line and determine if it could be used to select for the dormancy-capable subset of cells and to detect differences upon enrichment of dormant or chemoresistant cell populations. Upon encapsulation in silica gels, surviving OVCAR-3 cells demonstrated hallmarks of dormancy, including reduction in Ki67 expression, modulation of key cell cycle regulatory genes and dormancy-associated signaling pathways, and ability to resume proliferation when the stress of physical confinement was removed. Moreover, enrichment of the dormant subpopulation using hypoxia treatment or serum starvation prior to silica gel encapsulation resulted in enhanced survival in gels relative to cells grown in standard culture conditions. These results suggest that silica gel encapsulation may be used as a simple method to isolate dormancy-capable cells for further applications such as gene or protein expression analysis and drug screening. Similarly, cells already in a dormant state demonstrated lower susceptibility to cisplatin drug treatment relative to actively proliferating cells, further supporting the idea that dormant cancer cells are less susceptible to chemotherapy and that chemoresistance may stem from a subpopulation of dormant cancer cells. After extraction from silica gels and subsequent return to a proliferative state, cells surviving prolonged encapsulation also demonstrated enhanced survival after cisplatin treatment relative to control populations. This suggests that the dormancy-capable cells selected for within silica gels are indeed more chemoresistant, even when no longer dormant, and that the ability to enter dormancy and chemoresistance in ovarian cancer are likely somehow connected. Lastly, when the drug-resistant phenotype was selected for through cisplatin drug treatment prior to encapsulation, a significant difference in viability was observed upon encapsulation between cisplatin-treated and untreated cells. Overall, these results indicate that the silica gel technology and the behavior of ovarian cancer cells after encapsulation may be an indicator of cells inherently capable of entering dormancy and less susceptible to platinum-based chemotherapies. In future applications, silica gel encapsulation of patient ovarian cancer cells could potentially be used as a predictive clinical tool to identify patients at risk of early recurrence or as a facile in vitro platform to investigate mechanistic links between dormancy, chemoresistance, and recurrence. Citation Format: Tiffany Lam, Hak Rae Lee, Melissa A. Geller, Alptekin Aksan, Samira Azarin. SILICA GEL ENCAPSULATION AS A POTENTIAL TOOL TO IDENTIFY DORMANT AND DRUG-RESISTANT CELL POPULATIONS IN OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-008.

Abstract 2533: Regulation of proliferation and metastasis gene expression by DAX-1 (NR0B1) in human breast cancer cells

Abstract The orphan nuclear receptor DAX-1 (Dosage Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, critical region on the X chromosome, gene 1) is known to serve an important role early on in mammalian sex determination as well as in the regulation of expression of steroid hormones. While it is unclear whether DAX-1 functions independently, DAX-1 has been demonstrated to act as a repressor of many nuclear hormone receptors, including ER, AR, PR, LRH-1 and SF-1. DAX-1 plays a role in select adult tissues, including gonadal tissue. However, it has also been shown to play a role in several types of cancer, though its specific function is not well understood. For example, in some types of cancer DAX-1 expression is upregulated, while in other cancers it is highly downregulated or completely absent. In an effort to better understand DAX-1 function both in normal and disease states, we examined the actions of DAX-1 in MCF7 breast cancer cells, which do not express the DAX-1 gene. We probed three different PCR arrays (breast cancer genes, proliferation genes and metastatic genes) to determine which genes were influenced by DAX-1 expression. With the introduction of DAX-1 in ERα positive breast cancer cells (MCF-7), we find that DAX-1 inhibits cell proliferation and blocks estradiol activation of key cell cycle regulatory genes, such as cyclin D1, as well as important metastatic genes. We show that DAX-1 binds directly to ERα and the ERα-DAX-1 complex binds to the cyclin D1 promoter. The outcome of DAX-1 expression in MCF7 cells is cell cycle arrest through the repression of key cell cycle regulatory genes. These results demonstrate that DAX-1 functions as a corepressor of ERα in MCF7 breast cancer cells by preventing the activation of growth-promoting genes and decreasing cell proliferation in response to estrogens. These findings could have significant implications for future drug development strategies specifically aimed at ER-interacting proteins such as DAX-1. Citation Format: Christina Tzagarakis-Foster. Regulation of proliferation and metastasis gene expression by DAX-1 (NR0B1) in human breast cancer cells [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 2533.

Regulation of Mesenchymal Stem to Transit-Amplifying Cell Transition in the Continuously Growing Mouse Incisor

SummaryIn adult tissues and organs with high turnover rates, the generation of transit-amplifying cell (TAC) populations from self-renewing stem cells drives cell replacement. The role of stem cells is to provide a renewable source of cells that give rise to TACs to provide the cell numbers that are necessary for cell differentiation. Regulation of the formation of TACs is thus fundamental to controlling cell replacement. Here, we analyze the properties of a population of mesenchymal TACs in the continuously growing mouse incisor to identify key components of the molecular regulation that drives proliferation. We show that the polycomb repressive complex 1 acts as a global regulator of the TAC phenotype by its direct action on the expression of key cell-cycle regulatory genes and by regulating Wnt/β-catenin-signaling activity. We also identify an essential requirement for TACs in maintaining mesenchymal stem cells, which is indicative of a positive feedback mechanism.

Hunk, a Serine/Threonine Protein Kinase, Regulates Insulin Secretion from Pancreatic Islets

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder caused by the failure of the pancreatic β‐cells to secrete sufficient insulin to maintain glucose homeostasis. Genetic factors make a strong contribution to the development of the T2DM. Therefore, identifying and characterizing novel genetic factors that play an important role in β‐cell function is critical to understand the heritability of T2DM. We have recently identified the Hunk (Hormonally Up‐Regulated Neu‐Associated Kinase) gene as a novel regulator of insulin secretion from a mouse genetic screen. Genetic variation near the HUNK gene is associated with T2DM in humans. However, no study has yet been reported on the role of Hunk in pancreatic β‐cells.We studied the role of Hunk in two model systems; in islets isolated from whole‐body Hunk knockout mice (HunkKO), and in INS1 cells, a rat insulinoma β‐cell line. We observed that compared to wild type (WT) mice, insulin secretion in response to several different secretagogues was elevated in islets from HunkKO mice. This difference in the secretory response between WT and HunkKO islets was larger if the mice were maintained on a western‐style high‐fat/high‐sucrose (HF/HS) diet, suggesting that Hunk may be required for the diet to exert a suppressive effect on β‐cell function.In contrast to our observations with WT versus HunkKO islets, we observed a very different result in INS1 β‐cells. We transiently overexpressed control Hunk (Hunkcontrol), a catalytically inactive mutant of Hunk that contains a point mutation in the kinase domain (HunkCi), or GFP in INS1 β‐cells; 48 hr later we measured insulin secretion in response to different segretagogues in all three groups of cells. Compared to GFP, cells expressing Hunkcontrol, but not HunkCi, secreted more insulin in response to glucose alone, or a combination of glucose plus fatty acid. This result suggests that the augmented secretory response by Hunkcontrol required Hunk's catalytic activity.Hunk is known to regulate cellular proliferation in other tissues; e.g., breast cancer. We asked if Hunk controls cellular proliferation in pancreatic islets. β‐cells in adult islets can be induced to proliferate in response to metabolic stress, such as high fat diet. We measured the level of expression for several key cell cycle regulatory genes in islets isolated from WT and HunkKO mice that were maintained on the HF/HS diet for 4 months. In addition, we used the insulin receptor antagonist, S961, to induce a very high degree of insulin resistance that has previously been shown to result in significant β‐cell proliferation. Following one week of S961 infusion, islets were isolated and the expression levels of the cell cycle transcripts were measured. Compared to WT mice, cell cycle expression was not altered in the HunkKO islets when mice maintained on either the HF/HS diet, or when treated with S961. These results suggest that Hunk does not regulate β‐cell proliferation in response to metabolic stress.ConclusionWe identified Hunk, a Serine/Threonine protein kinase as a novel regulator of insulin secretion from beta cells. We find that for this particular gene, overexpression in a cell line does not yield a result predicted from a knockout in the mouse.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Histone Deacetylase 10 Regulates the Cell Cycle G2/M Phase Transition via a Novel Let-7-HMGA2-Cyclin A2 Pathway.

Histone deacetylase (HDAC) inhibition leads to cell cycle arrest in G1 and G2, suggesting HDACs as therapeutic targets for cancer and diseases linked to abnormal cell growth and proliferation. Many HDACs are transcriptional repressors. Some may alter cell cycle progression by deacetylating histones and repressing transcription of key cell cycle regulatory genes. Here, we report that HDAC10 regulates the cell cycle via modulation of cyclin A2 expression, and cyclin A2 overexpression rescues HDAC10 knockdown-induced G2/M transition arrest. HDAC10 regulates cyclin A2 expression by deacetylating histones near the let-7 promoter, thereby repressing transcription. In HDAC10 knockdown cells, let-7f and microRNA 98 (miR-98) were upregulated and the let-7 family target, HMGA2, was downregulated. HMGA2 loss resulted in enrichment of the transcriptional repressor E4F at the cyclin A2 promoter. These findings support a role for HDACs in cell cycle regulation, reveal a novel mechanism of HDAC10 action, and extend the potential of HDACs as targets in diseases of cell cycle dysregulation.

Novel Evidence for Curcumin and Boswellic Acid-Induced Chemoprevention through Regulation of miR-34a and miR-27a in Colorectal Cancer.

Colorectal cancer is one of the most common causes of cancer-associated mortality worldwide, but it is truly a preventable disease. Both curcumin and boswellic acids are well-established dietary botanicals with potent antitumorigenic properties that have been shown to modulate multiple oncogenic pathways. Recent data suggest that the chemopreventive effects of these botanicals may, in part, be mediated through regulation of key cancer-related microRNAs (miRNA) and their downstream gene targets. Here, we investigated the antitumorigenic effects of curcumin and 3 acetyl-11-keto-β-boswellic acid (AKBA) on modulation of specific cancer-related miRNAs in colorectal cancer cells and validated their protective effects in vivo using a xenograft mouse model. Both curcumin and AKBA inhibited cellular proliferation, induced apoptosis and cell-cycle arrest in colorectal cancer cell lines, and these effects were significantly enhanced with combined treatment. Gene-expression arrays revealed that curcumin and AKBA regulated distinct cancer signaling pathways, including key cell-cycle regulatory genes. Combined bioinformatics and in silico analysis identified apoptosis, proliferation, and cell-cycle regulatory signaling pathways as key modulators of curcumin and AKBA-induced anticancer effects. We discovered that curcumin and AKBA induced upregulation of tumor-suppressive miR-34a and downregulation of miR-27a in colorectal cancer cells. Furthermore, we demonstrated in a mouse xenograft model that both curcumin and AKBA treatments suppressed tumor growth, which corresponded with alterations in the expression of miR-34a and miR-27a, consistent with our in vitro findings. Herein, we provide novel mechanistic evidence for the chemopreventive effects of curcumin and AKBA through regulation of specific miRNAs in colorectal cancer.

Abstract 3115: Role of nuclear coactivator AIB1 in endothelial cell function and angiogenesis

Abstract The nuclear receptor coactivator oncogene AIB1 (Amplified In Breast Cancer 1) has a clearly defined role in steroid and growth factor signaling in epithelial cells of cancers. Recently, we have demonstrated that AIB1 is functionally required for HER2-mediated mammary tumorigenesis in MMTV/Neu mouse model for breast cancer through regulation of HER2 phosphorylation and signaling. Generation of neu/HER2 transgenic mice with either loss of one or both copies of the AIB1 gene resulted in reduced or complete abolition of mammary tumor development, respectively. One very interesting aspect of this study was that the tumors that arose in HER2 AIB1 HET (+/-) mice were phenotypically different from those in HER2 AIB1 WT (+/+) mice in that they were smaller, more necrotic and significantly less vascularized. This data indicated to us the possibility that the role of AIB1 was not confined to the mammary epithelium proliferation but also had major effects on the surrounding stromal cells. In addition, recent expression array data indicated that AIB1 is also overexpressed in human breast cancer stroma which includes the endothelium. In here, we examined the role of AIB1 in endothelial cell function. We demonstrate that reduction of AIB1 levels in endothelial cells cultured in vitro prevents their ability to form endothelial tubes, in conjuction with reduced proliferation and motility. Furthermore, in ECIS (Electric Cell-substrate Impedance Sensing) assay, HUVEC cells with low AIB1 expression loss the ability to form a monolayer on the surface of gold-film electrodes. Consistent with the reduced proliferation that we observed, cDNA array analysis that compared gene expression patterns of HUVEC cells +/- AIB1 shRNA indicates that a number of key cell cycle regulatory genes and cytokines were regulated by reduction in AIB1 levels. Overall these data describe a new role for nuclear receptor coactivator AIB1 in the endothelium. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3115. doi:10.1158/1538-7445.AM2011-3115

Regulation of apoptosis-associated genes by histone deacetylase inhibitors: implications in cancer therapy

Anomalous gene regulation, dictated by epigenetic modifications, is a universal characteristic of cancer cells. Histone deacetylases (HDACs) are an important class of enzymes that influence gene expression by the removal of acetyl groups from histones leading to chromatin remodeling and transcriptional suppression of key apoptosis and cell cycle regulatory genes. Histone deacetylase inhibitors (HDACis) are a novel category of anticancer pharmacological agents developed to counter the actions of HDACs, thus, inducing an array of cellular consequences, such as apoptosis, cell cycle arrest, generation of reactive oxygen species, inhibition of angiogenesis, and autophagy. Suberoylanilide hydroxamic acid (SAHA, Zolinza, Vorinostat), is currently the only Food and Drug Administration-approved HDACi for the treatment of cutaneous T-cell lymphoma. SAHA and other HDACis have shown selective toxicity toward malignant cells while sparing the surrounding normal cells. In addition to this specificity, their regulation of apoptosis-associated genes and the synergistic augmentation of apoptotic events when used simultaneously with other anticancer agents such as conventional chemotherapies, radiation, inhibitors of DNA methylation, and proteasome inhibitors make HDACis potential novel arsenals in the battle against cancer. Herein I review epigenetic modifications, discuss the various mechanisms of HDACi-induced effects, in particular modulation of expression of apoptosis-associated gene products, and highlight SAHA and its antitumor functions.

Differential Antineoplastic Effects of Butyrate in Cells With and Without a Functioning DNA Mismatch Repair

The aim of this study was to investigate the differential antineoplastic effects of butyrate in cells with and without a functional mismatch repair and to determine the molecular mechanisms underlying these effects. SW48 colon cancer cells in which the MLH1 gene is silenced by promoter hypermethylation and demethylated SW48 cells in which the MLH1 gene is reexpressed were treated with butyrate (0-5mM) for 8 days and the effects on cell number, MLH1 gene promoter methylation, and expression of two cell cycle regulatory genes, CDK4 and GADD45A, were assessed. Butyrate suppressed viable cell number ( P < 0.001) and reduced MLH1 promoter methylation ( P < 0.05) in SW48 cells. However, in demethylated SW48 cells, butyrate caused an increase in viable cells ( P < 0.05) and promoter methylation ( P < 0.05). CDK4 expression was downregulated by butyrate exposure, but the effect was significantly greater for demethylated SW48 cells ( P = 0.025). Butyrate treatment caused upregulation of GADD45A expression in SW48 cells but downregulation of GADD45A expression in demethylated SW48 cells ( P = 0.045). This study supports the hypothesis that butyrate has more potent antineoplastic effects on colon cancer cells with MLH1 dysfunction. Differential expression of key cell cycle regulatory genes may explain some of the molecular mechanisms underlying these effects.

Cell kinetics and gene expression changes in colorectal cancer patients given resistant starch: a randomised controlled trial

Objective: This study investigated the effects of oral supplementation of resistant starch (RS) on tumour cell and colonic mucosal cell kinetics and on gene expression in patients with colorectal cancer...

NF-Y Dependent Epigenetic Modifications Discriminate between Proliferating and Postmitotic Tissue

The regulation of gene transcription requires posttranslational modifications of histones that, in concert with chromatin remodeling factors, shape the structure of chromatin. It is currently under intense investigation how this structure is modulated, in particular in the context of proliferation and differentiation. Compelling evidence suggests that the transcription factor NF-Y acts as a master regulator of cell cycle progression, activating the transcription of many cell cycle regulatory genes. However, the underlying molecular mechanisms are not yet completely understood. Here we show that NF-Y exerts its effect on transcription through the modulation of the histone “code”. NF-Y colocalizes with nascent RNA, while RNA polymerase II is I phosphorylated on serine 2 of the YSPTSPS repeats within its carboxyterminal domain and histones are carrying modifications that represent activation signals of gene expression (H3K9ac and PAN-H4ac). Comparing postmitotic muscle tissue from normal mice and proliferating muscles from mdx mice, we demonstrate by chromatin immunoprecipitation (ChIP) that NF-Y DNA binding activity correlates with the accumulation of acetylated histones H3 and H4 on promoters of key cell cycle regulatory genes, and with their active transcription. Accordingly, p300 is recruited onto the chromatin of NF-Y target genes in a NF-Y-dependent manner, as demonstrated by Re-ChIP. Conversely, the loss of NF-Y binding correlates with a decrease of acetylated histones, the recruitment of HDAC1, and a repressed heterochromatic state with enrichment of histones carrying modifications known to mediate silencing of gene expression (H3K9me3, H3K27me2 and H4K20me3). As a consequence, NF-Y target genes are downregulated in this context. In conclusion, our data indicate a role of NF-Y in modulating the structure and transcriptional competence of chromatin in vivo and support a model in which NF-Y-dependent histone “code” changes contribute to the proper discrimination between proliferating and postmitotic cells in vivo and in vitro.

Steroidal regulation of cell cycle progression.

Sex steroid hormones and their antagonists have well-defined mitogenic and growth-inhibitory effects on target cells including cancer cells. These effects are mediated by cell cycle phase-specific actions, implying that steroids control rates of cell cycle progression by regulating the expression of key cell cycle regulatory genes. An emerging model of cell cycle control involves transcriptional induction of cyclin genes and consequent activation of cyclin-dependent kinases, which initiate cellular events necessary to complete checkpoints within the cell cycle. Our recent studies have focused on the roles of G1 cyclins, particularly cyclin D1, in the control of cell cycle progression in human breast cancer cells. These studies show that cyclin D1 induction is an early response to mitogenic stimulation by oestrogens and progestins, is rate-limiting for G1 progression and is sufficient for completion of the cell cycle in cells arrested in early G1 phase by serum deprivation. Furthermore, inhibition of cyclin D1 expression is an early response to growth-inhibitory anti-oestrogens. These results suggest that cyclin D1 is a target for regulation of cell cycle progression by sex steroids and their antagonists.

Role of Resistant Starch in Colorectal Cancer Prevention: A Prospective Randomised Controlled Trial

Purpose: Colorectal cancer (CRC) is the second most common cause of cancer related death in the western world. Resistant starch (RS), defined as starch that resists digestion in the small intestine of a healthy individual, gets converted to butyrate following bacterial fermentation in the colon. Butyrate is shown to have potent anti-neoplastic effects on colon cancer cells in vitro. This study investigated the anti-neoplastic effects of RS in patients with CRC and its potential role of as a chemo-preventive agent. Methods: A total of 65 (36 male) sporadic CRC patients were randomised to treatment (40 gm/ day) with RS or ordinary starch (OS) for 2–4 weeks. Pre-treatment and post-treatment biopsies were obtained from tumour and normal mucosa and the effects of the intervention on cell proliferation and expression of cell cycle regulatory genes CDK4 and GADD45A (using real time RT-PCR) were investigated. Results: The proportion of mitotic cells in the top half of the crypt (which is a valid pre-malignant marker used to assess response to chemo-preventive trials) was markedly reduced following RS treatment as compared with OS treatment (P= 0.028) (Fig. 1). There was no effect of RS treatment on crypt dimensions or tumour proliferation index. The expression of key cell cycle regulatory gene CDK4 was upregulated (P < 0.01) while that of GADD45A was down regulated (P < 0.001) in the tumour tissue compared with normal flat mucosa. RS treatment for up to 4 weeks in CRC patients tended to increase CDK4 expression (P= 0.07) in tumour tissue. Expression of GADD45A, which was suppressed in cancer, was significantly upregulated (P= 0.048) following RS treatment (Fig. 2). Conclusion: RS modulates the colonic crypt cell kinetics and has potential as a chemo-preventive agent against CRC. The differential expression of key cell cycle regulatory genes may play a role in these cellular effects of RS.[figure1]Figure

Promoter hypermethylation profile of cell cycle regulator genes in pituitary adenomas

Aberrant hypermethylation of CpG islands in the promoter region plays a causal role in the inactivation of various key genes involved in the cell cycle regulatory cascade, which could result in a loss of cell cycle control. The aim of the present study was to examine in more detail the prevalence and role of the promoter methylation of genes with a proven involvement in the cell cycle regulation of pituitary adenomas, since their tumorigenesis has not yet been clearly defined. We profiled the CpG island methylation status of a series of well-characterized cell cycle regulation genes: the RB1, p14(ARF), p15(INK4b), p16(INK4a), p21(Waf1/Cip1), p27(Kip1), and p73 genes, in 34 pituitary adenomas as determined by a methylation-specific polymerase chain reaction assay. Promoter hypermethylation of the RB1, p14(ARF), p15(INK4b), p16(INK4a), p21(Waf1/Cip1), p27(Kip1), and p73 genes was detected in 12 (35%), 2 (6%), 11 (32%), 20 (59%), 1 (3%), 0 (0%), and 4 (12%) of the adenomas, respectively. In total, 88% (30 of 34) of the adenomas displayed methylation of at least one of such cell cycle regulatory genes, especially methylation of the member genes of the RB1 pathway (29 of 34; 85%). Promoter hypermethylation of p15(INK4b) coincided with RB1 and/or p16(INK4a) methylation, whereas RB1 and p16(INK4a) methylations tended to be mutually exclusive (p = 0.0048). Furthermore, promoter hypermethylations of p14(ARF), p21(Waf1/Cip1), and p73 (not belonging to the member genes of the RB1 pathway) were also coincident with RB1 and/or p16(INK4a) methylation except in one p73 methylated case. In contrast, none of the clinicopathological features, including the cell proliferation index, was significantly correlated with any particular methylation status. Our results suggested that aberrant hypermethylation of the key cell cycle regulatory genes occurs at a relatively high frequency in pituitary adenomas, especially in RB1 pathway genes with promoter hypermethylation of the p16(INK4a) gene being the most common deregulation. We further obtained evidence to indicate that RB1 and p16(INK4a) methylations tended to be mutually exclusive, but did occasionally coincide with other cell cycle regulation gene methylations.

Status of Chromosome 13 in Multiple Myeloma: Integrated Approach Using SNP Mapping Array and Gene Expression Array.

Deletions on chromosome 13 are thought to be one of the most important prognostic features in Multiple Myeloma (MM). The biology underlying this is, however, uncertain. Chromosome 13 abnormalities have been evaluated conventionally by FISH using probes for 13q14, covering the retinoblastoma gene (RB1) region. Typically, for recurrent regions of loss of heterozygosity (LOH) it is possible to map a minimally deleted region within which an important gene may be located. This should be the case with 13q−, or alternatively there may be linkage with another genetic lesion, which could be contributing to the poor prognosis. Following the implementation of high-density single-nucleotide polymorphism (SNP) array, it is now possible to genotype the whole human genome with a mapping resolution of less than 50 Kb. Thus, the SNP array approach offers an opportunity to analyze both copy number abnormalities and LOH simultaneously. The aim of this study was to determine the numerical alterations, LOH and changes in the gene expression profile of the chromosome 13 in MM, and its possible association with other genetic events. For this purpose, we analyzed 17 patients included on the Myeloma IX trial with deletion of 13q14 compared with 22 samples without deletion, using Affymetrix 50K SNP arrays and Affymetrix U133 Plus 2 expression array. IGH translocations and 13q deletion were determined by FISH. dChipSNP and WGSA programs were used to analyze the data. With respect to 13q14, there was 100% correlation between FISH and SNP array results. 16 out of 17 cases with deletion of the RB1 gene by FISH analysis showed loss of 13q arm by SNP array, demonstrating that loss of the whole chromosome 13 is responsible for 13q deletions found in MM in >90% of cases, with only one case showing a defined region of deletion of chromosome 13 (13q14.11–13q21.2). Using gene expression arrays we could not define a specific pattern characteristic of expression loss in genes at 13q. Lower RB1 expression levels were not only restricted to cases with del(13). However, samples containing IGH translocations (t(11;14) and t(4;14)) without del(13) showed up to 4 times more RB1 expression, suggesting that MM evolution in cases containing IGH translocations is independent of RB1 expression. Interestingly, the hyperdiploid cases with and without del(13) expressed similar level of RB1. We also investigated whether other key cell cycle regulatory genes were associated with del(13); in particular, 4 cases showed 9p21 LOH by SNP array and no different gene expression levels, which suggest that LOH does not seem to be a mechanism of lost of expression of CDKN2A, CDKN2B and p14/ARF. We could not find any significant correlation with del(13) and expression of cell cycle regulatory genes, apart from 8/17 samples with del(13) that had low expression of p53 gene, including 6 t(4;14) cases and 2 t(11;14) cases. Also, 2 cases without monosomy 13 (1 with t(4;14) and 1 with t(11;14)), showed low p53 expression levels. However, SNP array data did not show any deletion at 17p in 38 cases, with the exception of a case with monosomy 13 and t(11;14) in which SNP array data showed loss at 17pter-17q21.2 and FISH detected p53 deletion. Further investigation between the association of p53 and del(13) are ongoing and maybe useful in defining the biology of this poor subgroup of patients.

Suppression of human prostate cancer cell growth by ciprofloxacin is associated with cell cycle arrest and apoptosis

For hormone resistant prostate cancer (HRPC), chemotherapy is used but the mortality is 100% with a mean survival time of 7-8 months. Our previous studies have shown the chemotherapeutic effect of ciprofloxacin in bladder cancer. At doses 50-400 micro g/ml ciprofloxacin, the concentrations that are normally achieved at doses currently used for the treatment of anti-bacterial infections, inhibited bladder cancer cell growth and induced S/G2M arrest with modulation of key cell cycle regulatory genes and ultimately activated apoptotic processes. In this study, we investigated the effect of ciprofloxacin on androgen independent prostate carcinoma, PC3 cells and compared our results with non-tumorigenic prostate epithelial cells. The main advantage of this fluroquinolone antibiotic is its relative non-toxicity as compared to current chemotherapy, which is not very effective, for the treatment of advanced hormone resistant prostate cancer. PC3 cells as well as normal prostate epithelial cells (MLC8891) were treated with 25-400 micro g/ml ciprofloxacin, and cell counting was done during 3 days of treatment. The cell death was determined using DAPI staining of cell nuclei, 7AAD-staining followed by flow cytometric analysis as well as by activation of caspase-3, a member of the ICE family of enzymes involved in the apoptotic cascade. The cell lysates were analyzed by immunoblotting techniques for the expression of key genes targeted by ciprofloxacin (p21WAF1, Bax and Bcl-2). Translocation of bax was visualized using a fluorescence staining procedure followed by laser confocal microscopic imaging. Treatment of prostate cancer cells with ciprofloxacin resulted in a dose- and time-dependent inhibition of cell growth (70-100% with 50-400 micro g/ml of the drug). There was a concomitant induction of cell cycle arrest at the S and G2/M phases of the cell cycle as well as induction of apoptosis. The CDK inhibitor p21WAF1 was down-regulated as early as 12 h following ciprofloxacin treatment (100-200 micro g/ml for 12-24 h). There was a significant increase in the Bax/Bcl-2 ratio with translocation of Bax, a pro-apoptotic protein, to mitochondria with concomitant activation of caspase 3. These results suggest the potential usefulness of the fluroquinolone, ciprofloxacin as a chemotherapeutic agent for advanced prostate cancer. The fluroquinolone ciprofloxacin showed anti-proliferative and apoptosis inducing activity on prostate cancer cells but not on non-tumorigenic prostate epithelial cells. These effects of ciprofloxacin were mediated by cell cycle arrest at S-G2/M phase of the cell cycle, Bax translocation to mitochondrial membrane and by increasing the Bax/Bcl-2 ratio in PC3 prostate cancer cells. Based on our in vitro results, further in-depth in vivo animal or human investigations are warranted.

Developmental control of the cell cycle.

Whereas animal development has a defined and early embryonic phase during which all of the major organ systems are put in place, plant growth continues throughout the life of the organism. Moreover, most of the above ground plant growth that we see is modular and the modules, known as phytomers, are formed by the activity of shoot apical meristem. This results in at least two distinct types of growth: indeterminate growth as shown by the meristem, producing tissues of undefined maximum size; and determinate growth as shown by leaves and other lateral organs, producing organs of limited maximum size. We are interested in how plant cell proliferation is regulated within the context of development. Mutations in developmental genes can affect the pattern and the extent of cell growth or proliferation. Thus it can be argued that such genes regulate cell growth division. The molecular mechanisms by which developmental genes influence cell division and proliferation have remained largely obscure. Developmental patterning and cell cycle control appear to be co-ordinated throughout plant growth and this is reflected by temporal and spatial regulation of regulatory genes such as cyclins and cyclin dependent protein kinases. Using an approach based on RNA in situ hybridisation, we have characterised the expression patterns of a number of cell cycle genes (Fobert et al., 1994; Fobert et al., 1996; Gaudin et al., 2000). Based on this survey, many cell cycle control genes are expressed throughout the plant in a cell cycle-stage specific manner, and produce a spotty pattern (Fig. 1). Double label in situ methods (Fobert et al., 1994) and synchronised cell (Sorrell et al., 2001) studies permit the further resolution of expression periods (Fig. 1). Many of the genes, such as B-cyclins, kinesins and ICKs expressed during the G2/M phase, contain characteristic motifs, called MSA elements, within their 5# non-coding regions. Molecular analysis of these MSA elements indicates that they are necessary and sufficient to direct G2/M gene expression and that they bind to 3 repeat Myb proteins (Ito et al., 2001). The plant 3 repeat Myb proteins are similar to the mammalian cMybs that are implicated in G1/S progression. Therefore, their role has been conserved in that both animal and plant genes regulate cell cycle phase transitions, but the phase affected is different. Plant 3 repeat Myb genes form two classes, one of which appears to act positively and is up regulated during G2/M; the other acts as an inhibitor and is expressed throughout the cell cycle. The transcripts of other cell cycle related genes do not change markedly during the cell cycle. These include the PSTAIR cdk genes and cyclin A and D genes. These functions may be regulated post-transcriptionally. Gainand loss-of-function transgenic and mutant approaches are being used to dissect the function of such genes. The alcA/alcR gene switch is a two component chemically inducible system for regulating gene expression in a range of organisms. Briefly, this system involves two components, originally from the filamentous fungus, Aspergillus nidulans. The transactivator, ALCR, is activated by a treatment of the plant with a low level of ethanol and a chimeric promoter, pAlcA/35S that contains a minimal 35S promoter and the upstream regulatory sequences of the Aspergillus alcA gene containing the binding sites of ALCR. The ethanol switch is functional in tobacco and has been transferred to Arabidopsis (Roslan et al., 2001) where we are using it to up and down regulate the expression of key developmental and cell cycle regulatory genes. We have created a range of transactivator lines that express the alcR transactivator in specific domains of the meristem or leaves. These lines should be generally useful for manipulating meristem growth or development. * Corresponding author. Tel.: +44-1603-450288; fax: +44-1603-450022. E-mail address: john.doonan@bbsrc.ac.uk (J.H. Doonan). Cell Biology International 27 (2003) 283–285 Cell Biology International

E2F4 loss suppresses tumorigenesis in Rb mutant mice

The E2F transcription factors mediate the activation or repression of key cell cycle regulatory genes under the control of the retinoblastoma protein (pRB) tumor suppressor and its relatives, p107 and p130. Here we investigate how E2F4, the major “repressive” E2F, contributes to pRB's tumor-suppressive properties. Remarkably, E2F4 loss suppresses the development of both pituitary and thyroid tumors in Rb +/− mice. Importantly, E2F4 loss also suppresses the inappropriate gene expression and proliferation of pRB-deficient cells. Biochemical analyses suggest that this tumor suppression occurs via a novel mechanism: E2F4 loss allows p107 and p130 to regulate the pRB-specific, activator E2Fs. We also detect these novel E2F complexes in pRB-deficient cells, suggesting that they play a significant role in the regulation of tumorigenesis in vivo.

Aberrant p16(INK4A) and DPC4/Smad4 expression in intraductal papillary mucinous tumours of the pancreas is associated with invasive ductal adenocarcinoma.

Intraductal papillary mucinous tumours (IPMT) of the pancreas constitute a unique pathological entity with an overall incidence of associated invasive malignancy of 20%. The malignant potential of an individual IPMT cannot be accurately predicted. Preoperative estimation of the risk of associated invasive malignancy with IPMT would be of significant clinical benefit. As aberrations in cell cycle regulatory genes are associated with the progression of precursor pancreatic ductal lesions to invasive adenocarcinoma, we examined expression of key cell cycle regulatory genes in the cyclin D1/retinoblastoma pathway and the transforming growth factor beta/Smad4 signalling pathway in a cohort of patients with surgically resected IPMT. Sections of formalin fixed paraffin embedded pancreatic tissue from a cohort of 18 patients with IPMT were examined using immunohistochemistry for protein expression of cell cycle regulatory genes p16(INK4A), p21(CIP1), p27(KIP1), cyclin D1, pRb, and p53, as well as the cell signalling molecule Smad4. A comparison of expression levels was made between adenoma/borderline IPMT (10 patients) and intraductal papillary mucinous carcinoma (IPMC) (eight patients, four of whom harboured invasive carcinoma). Statistical analysis was performed using the chi(2) and Fisher's exact tests. Aberrant expression of the proteins examined increased in frequency from adenoma/borderline IPMT to IPMC. Specifically, there was a significantly greater incidence of loss of p16(INK4A) expression in IPMC: 8/8 lesions (100%) compared with 1/10 (10%) adenoma/borderline IPMT (p<0.001). Similarly, loss of Smad4 expression was associated with IPMC: 3/8 (38%) versus adenoma/borderline IPMT 0/10 (p<0.03). Loss of Smad4 expression within the IPMT was the best marker for the presence of invasive carcinoma (p<0.001). These data indicate that loss of p16(INK4A) and Smad4 expression occur more frequently in IPMC alone, or with associated invasive carcinoma, compared with adenoma/borderline IPMT. Aberrant protein expression of these cell cycle regulatory genes in IPMT and pancreatic intraepithelial neoplasia in the current model of pancreatic cancer progression suggest similarities in their development and may also represent the subsequent risk of invasive carcinoma.