Cell Cycle Control Genes Research Articles

Novel mithramycins abrogate the involvement of protein factors in the transcription of cell cycle control genes

The effects of mithramycin SK (MSK) and demycarosyl-3D-β-d-digitoxosyl-mithramycin SK (DIG-MSK; EC-8042), two novel analogs of the antitumor antibiotic mithramycin A, on gene transcription were examined in human HCT116 colon carcinoma cells by quantitative real-time PCR of 89 genes mainly involved in cell cycle control. Each one of the analogs down-regulated a different set of genes, while only five genes were down-regulated by both compounds. Moreover, other genes were significantly up-regulated, among them p21WAF1/CDKN1A which is involved in halting cells at the G1 and G2/M checkpoints. These results are rationalized in terms of MSK or DIG-MSK competition with various transcription factors for binding to consensus C/G-rich tracts encompassed in gene promoters. Changes in cell cycle distribution and protein levels after treatment with every analog were consistent with changes observed in gene expression.

IGH@ Translocations, CRLF2 Deregulation, and Microdeletions in Adolescents and Adults With Acute Lymphoblastic Leukemia

To determine the prevalence and prognostic impact of significant acute lymphoblastic leukemia (ALL) -related genes: CRLF2 deregulation (CRLF2-d), IGH@ translocations (IGH@-t), and deletions of CDKN2A/B, IKZF1, PAX5, ETV6, RB1, BTG1, and EBF1 in adolescents and adults. The cohort comprised 454 patients (age 15 to 60 years old) treated on the multicenter United Kingdom Acute Lymphoblastic Leukaemia Trial XII/Eastern Cooperative Oncology Group 2993 trial (UKALLXII/ECOG2993) with Philadelphia-negative B-cell precursor ALL. Fluorescent in situ hybridization and multiplex ligation-dependent probe amplification were used to detect these genetic alterations. Twenty patients (5%) had CRLF2-d (P2RY8-CRLF2, n = 7; IGH@-CRLF2, n = 13), and 36 patients (8%) harbored an IGH@-t with a different partner gene. There was little overlap between IGH@-t, CRLF2-d, and established chromosomal abnormalities. Deletions of CDKN2A/B, IKZF1, PAX5, ETV6, RB1, BTG1, or EBF1 were prevalent with 101 (33%) of 304 patients harboring one and 102 (33%) harboring two or more alterations, occurring with varying frequency in all cytogenetic subgroups. The 5-year event-free survival, relapse-free survival (RFS), and overall survival (OS) rates for the whole cohort were 40%, 55%, and 43%, respectively. Patients with CRLF2-d, IGH@-t, and IKZF1 deletions were associated with an inferior outcome in univariate but not multivariate analysis. In particular, CRLF2-d patients had a lower RFS compared with other patients (30%), whereas those with IGH@-t or IKZF1 deletions had a lower OS (27% and 35%, respectively). CRLF2-d and IGH@-t represent distinct subtypes of adolescent and adult ALL. Deletions of key B-cell differentiation and cell cycle control genes are highly prevalent but vary in frequency by cytogenetic subgroup. CRLF2-d, IGH@-t, and IKZF1 deletions are associated with poor outcome in adolescent and adult ALL.

Effects of epidermal growth factor on the proliferation and cell cycle regulation of cultured human amnion epithelial cells

Human amnion epithelial cells (HAECs) hold great promise in tissue engineering for regenerative medicine. Large numbers of HAECs are required for this purpose. Hence, exogenous growth factor is added to the culture medium to improve epithelial cells proliferation. The aim of the present study was to determine the effects of epidermal growth factor (EGF) on the proliferation and cell cycle regulation of cultured HAECs. HAECs at P1 were cultured for 7 days in medium containing an equal volume mix of HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of EGF (0, 5, 10, 20, 30 and 50 ng/ml EGF) in reduced serum. Morphology, growth kinetics and cell cycle analysis using flow cytometry were assessed. Quantitative gene expression for cell cycle control genes, pluripotent transcription factors, epithelial genes and neuronal genes were also determined. EGF enhanced HAECs proliferation with optimal concentration at 10 ng/ml EGF. EGF significantly increased the proportion of HAECs at S- and G2/M-phase of the cell cycle compared to the control. At the end of culture, HAECs remained as diploid cells under cell cycle analysis. EGF significantly decreased the mRNA expression of p21, pRb, p53 and GADD45 in cultured HAECs. EGF also significantly decreased the pluripotent genes expression: Oct-3/4, Sox2 and Nanog; epithelial genes expression: CK14, p63, CK1 and Involucrin; and neuronal gene expression: NSE, NF-M and MAP 2. The results suggested that EGF is a strong mitogen that promotes the proliferation of HAECs through cell cycle regulation. EGF did not promote HAECs differentiation or pluripotent genes expression.

Medullary Thyroid Carcinoma: Who's on First?

ThyroidVol. 22, No. 5 Editorials and CommentaryMedullary Thyroid Carcinoma: Who's on First?Mimi I. Hu and Gilbert J. CoteMimi I. HuSearch for more papers by this author and Gilbert J. CoteSearch for more papers by this authorPublished Online:30 Apr 2012https://doi.org/10.1089/thy.2012.2205.edAboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited bySmall-molecule inhibitors, immune checkpoint inhibitors, and more: FDA-approved novel therapeutic drugs for solid tumors from 1991 to 20218 October 2022 | Journal of Hematology & Oncology, Vol. 15, No. 1Hereditary and familial thyroid tumours14 December 2017 | Histopathology, Vol. 72, No. 1Noncoding RNA blockade of autophagy is therapeutic in medullary thyroid cancer8 December 2014 | Cancer Medicine, Vol. 4, No. 2Smoldering medullary thyroid carcinoma liver metastasis 37 years after resection of an organ-confined tumor18 February 2014 | Diagnostic Cytopathology, Vol. 43, No. 1Thyroid C-Cell Biology and Oncogenic Transformation23 October 2015Polymorphisms of cell cycle control genes influence the development of sporadic medullary thyroid carcinoma6 November 2014 | European Journal of Endocrinology, Vol. 171, No. 6Molecular Testing in Thyroid CancerThe Role of Cdk5 in Neuroendocrine Thyroid CancerCancer Cell, Vol. 24, No. 4Exomic Sequencing of Medullary Thyroid Cancer Reveals Dominant and Mutually Exclusive Oncogenic Mutations in RET and RASThe Journal of Clinical Endocrinology & Metabolism, Vol. 98, No. 2 Volume 22Issue 5May 2012 InformationCopyright 2012, Mary Ann Liebert, Inc.To cite this article:Mimi I. Hu and Gilbert J. Cote.Medullary Thyroid Carcinoma: Who's on First?.Thyroid.May 2012.451-453.http://doi.org/10.1089/thy.2012.2205.edPublished in Volume: 22 Issue 5: April 30, 2012PDF download

Abstract 2185: BRD4 is a novel therapeutic target in melanoma

Abstract The incidence of melanoma is increasing faster than that of any other cancer, and predicted to double every 10-20 years. Surgery can be curative in Stage I, II, or III disease, but 75% of patients with deep primary lesions develop extensive recurrence or distant metastases and have dismal prognosis. In fact, there is no curative treatment for stage IV melanoma. Although novel targeted therapies, such as BRAF inhibitors and anti-CTLA4 antibodies are showing promising results in melanoma clinical trials, resistance to these agents and patient relapse rapidly ensue. Therapeutic resistance has been commonly attributed to functional redundancy between intimately hardwired cellular pathways responsible for tumor cell maintenance and survival. In order to avoid redundancy, we propose to directly inhibit the transcription of multiple genes required for the establishment or maintenance of tumors. First, we analyzed the expression of Bromodomain (BrD)-containing proteins, a family of epigenetic readers that bind acetylated lysine. BrDs are present in histone acetyl transferases (i.e. CBP/p300, PCAF, GCN5) and transcriptional regulators (i.e. BET family members: BRD2, 3, and 4). mRNA expression arrays showed several BrD-containing genes as upregulated in primary and metastatic melanoma cell lines compared to normal melanocytes. Analysis of available expression profiles also revealed higher levels of BRD2 and BRD4 in melanoma tissues relative to nevi or normal skin (P<0.001). Furthermore, immunohistochemistry staining of a melanoma tissue microarray confirmed overexpression of BRD4 protein in primary (P<0.001) and metastatic tumors (P<0.001) compared to nevi. BRD4 knockdown using siRNA or shRNA suppressed the proliferation and colony formation capacity of several metastatic melanoma cell lines. Moreover, melanoma cells stably infected with shBRD4 carrying lentivirus displayed reduced tumor growth in vivo compared with their counterpart infected with a non-silencing control. To identify cellular pathways modulated by BrD inhibition and to investigate the mechanism(s) that might mediate its anti-proliferative effects, we conducted a global transcriptome analysis (RNA sequencing) of 6 melanoma cell lines treated with specific BrD inhibitors or vehicle. Gene ontology analysis of these data revealed enrichment in genes controlling cell cycle, growth and proliferation. Accordingly, cell cycle analysis demonstrated a specific G1 phase arrest upon BRD4 silencing. In conclusion, we have demonstrated that expression of several BrD containing proteins is dysregulated in melanoma and that BRD4 represents a promising therapeutic target in these tumors. Our data provides a rationale for testing the efficacy of newly developed, specific and potent BET inhibitors against melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2185. doi:1538-7445.AM2012-2185

Messenger RNA Expression and Genetic Polymorphisms of Cell Cycle Control Genes and Chromosomal Aberrations in Chinese Vinyl Chloride Monomer-Exposed Workers

Messenger RNA Expression and Genetic Polymorphisms of Cell Cycle Control Genes and Chromosomal Aberrations in Chinese Vinyl Chloride Monomer-Exposed Workers

CHEK2*1100delC and Risk of Malignant Melanoma: Danish and German Studies and Meta-Analysis

It is possible that reduced function of DNA repair and cell-cycle control genes increases the individual susceptibility to malignant melanoma. As CHEK2 is a cell-cycle master controller, we tested the hypothesis that heterozygosity for the frameshift alteration CHEK2*1100delC is associated with increased risk of malignant melanoma. First, we performed case-control studies of 1,152 Danish and 752 German individuals with malignant melanoma compared with 9,142 Danish and 3,718 German controls. Second, we performed a meta-analysis of CHEK2*1100delC and malignant melanoma, involving 2,619 cases and 17,481 controls. Third, we examined the risk of malignant melanoma associated with CHEK2*1100delC heterozygosity in an analysis stratified for sun exposure, as well as for subtype and location on the body. The odds ratios for malignant melanoma for CHEK2(*)1100del heterozygotes compared with those for noncarriers were 2.01 (95% confidence interval (CI), 1.03-3.91) in Danes, 1.42 (95% CI, 0.46-4.31) in Germans, and 1.79 (95% CI, 1.02-3.17) in Danes and Germans combined. In a meta-analysis, the odds ratio of malignant melanoma for CHEK2*1100delC heterozygotes compared with that for noncarriers was 1.81 (95% CI, 1.07-3.05). Stratifications did not alter these results. CHEK2*1100delC heterozygotes have a twofold risk of malignant melanoma compared with noncarriers.

Glucocorticoid alternative effects on proliferating and differentiated mammary epithelium are associated to opposite regulation of cell‐cycle inhibitor expression

Glucocorticoids influence post-natal mammary gland development by sequentially controlling cell proliferation, differentiation, and apoptosis. In the mammary gland, it has been demonstrated that glucocorticoid treatment inhibits epithelial apoptosis in post-lactating glands. In this study, our first goal was to identify new glucocorticoid target genes that could be involved in generating this effect. Expression profiling, by microarray analysis, revealed that expression of several cell-cycle control genes was altered by dexamethasone (DEX) treatment after lactation. Importantly, it was determined that not only the exogenous synthetic hormone, but also the endogenous glucocorticoids regulated the expression of these genes. Particularly, we found that the expression of cell cycle inhibitors p21CIP1, p18INK4c, and Atm was differentially regulated by glucocorticoids through the successive stages of mammary gland development. In undifferentiated cells, DEX treatment induced their expression and reduced cell proliferation, while in differentiated cells this hormone repressed expression of those cell cycle inhibitors and promoted survival. Therefore, differentiation status determined the effect of glucocorticoids on mammary cell fate. Particularly, we have determined that p21CIP1 inhibition would mediate the activity of these hormones in differentiated mammary cells because over-expression of this protein blocked DEX-induced apoptosis protection. Together, our data suggest that the multiple roles played by glucocorticoids in mammary gland development and function might be at least partially due to the alternative roles that these hormones play on the expression of cell cycle regulators.

Abstract P119: S100A6 Regulates Endothelial Cell Physiology

Endothelial cell dysfunction is central to the development of cardiovascular pathologies. While Calcium cycling is of recognized importance to myocyte physiology and cardiac perfomance, the role of Ca2+ signaling in endothelial cell (EC) physiology remains relatively unexplored. In this study we investigated the role of S100A6 (calcyclin), an EF-hand type Ca2+ -binding protein that modulates target protein function dependent upon intracellular Ca2+ signaling. S100A6 displays cytoplasmic distribution in quiescent, confluent HUVEC, but translocates to the nucleus upon serum stimulation. Oxidative stress, induced by incubation of HUVEC with H¬2O2 (150 uM) leads to redistribution of nuclear S100A6 to the cytosol and cell cycle arrest. Knockdown of S100A6 by siRNA transfection reduced S100A6 levels by over 70% and diminished DNA synthesis by more than 75% (±12%, n=9, p<0.01), as well as expression of the proliferation markers PCNA and KI-67 (by more than 70% (±15%, n=9, p<0.01). Reduced S100A6 levels in HUVEC lead to an increase in cellular senescence, as measured by the expression of senescence associated β-galactosidase expression (by more than 6-fold, n=4, p<0.01) and a reduction of EC tube formation on matrigel matrix (by more than 90%, n=3, p<0.01). Reduction of S100A6 increased the expression of the cell cycle control and DNA repair-associated gene BRCA2, but did not change in the expression of BRCA1, cyclins or p53 (by RT-PCR array). We conclude that Ca2+ regulation by S100A6 is essential for multiple aspects of EC physiology.

Messenger RNA Expression and Genetic Polymorphisms of Cell Cycle Control Genes and Chromosomal Aberrations in Chinese Vinyl Chloride Monomer-Exposed Workers

To evaluate the expressions of p53, p21, and CCND1 in the peripheral blood lymphocytes of vinyl chloride monomer (VCM)-exposed workers and potential relationships with their exposures, polymorphisms, and chromosomal aberrations. The study was performed on 77 occupationally VCM-exposed workers and 43 unexposed controls. The quantities of mRNA expression of p53, p21, and CCND1 genes were detected by real-time polymerase chain reaction. p53 mRNA expression of VCM-low- and high-exposure groups was significantly lower than that of nonexposed group (P < 0.001), but p21 mRNA expression of the two VCM-exposed groups was significantly higher than that of the nonexposed group (P < 0.001). This study did not find the relationship between chromosomal aberrations, genotypes, and the expression of p53, p21, and CCND1. Messenger RNA expressions of p53 and p21 are changed with VCM-exposure status.

Copy Number Alterations of PAX5, CDKN2A/B Genes Rather Than IKZF1 gene Are Associated with Pathogenesis of Childhood ALL in Korean Assessed by Multiplex Ligation-Dependent Probe Amplification

Abstract 4231 Background:Recent genomic studies have analyzed genetic alterations and gene expression in childhood acute lymphoblastic leukemia (ALL), and detected multiple new submicroscopic genetic abnormalities in key cellular pathways, and also identified new prognostic markers and therapeutic targets. More than 50 recurring genetic alterations have been identified, and these genes are known to play key or putative roles in lymphoid development and leukemogenesis. Multiplex Ligation-dependent Probe Amplification (MLPA) is a rapid multiplex PCR based technique that allows the comparative quantification of multiple sites. However, there have been no reports on the MLPA based copy number alterations (CNA) of childhood ALL in Asian countries. The aim of this study is to reveal the CNA of genes involved in lymphoid differentiation and cell cycle control in Korean children with ALL. Patients and Methods:Reference ranges for individual MLPA probes were established from a group of 30 healthy control subjects. The MLPA assay was performed for 121 children with ALL in Korea to target those genes including the lymphoid differentiation genes (BTG1, EBF1, IKZF1, and PAX); cell cycle control genes (CDKN2A/B and RB1); transcriptional factors regulating genes (ETV6); and those deleted from the PAR1 region (IL3RA, CRLF2 and CSF2RA). We also evaluated the prognostic impact of these abnormalities. Results:Out of the 121 patients analyzed in our study, 96 (79%) displayed CNA in at least one gene: PAX5, 49 (40%); CDKN2A, 42 (35%); CDKN2B, 35 (29%); IKZF1, 22 (18%); ETV6, 22 (18%); EBF1, 16 (13%); RB1, 16 (13%); BTG1, 14 (12%); CRLF2, 12 (10%); IL3RA, 12 (10%); CSF2RA, 11 (9%). Thirteen of 121 (11%) patients relapsed. There was no significant correlation between these CNA and the recurrence of ALL. Conclusion:These results suggest that CNA of PAX5, CDKN2A/B may play an important role in the pathogenesis of childhood ALL in Korea, contrasting to those findings from western countries showing a significant correlation between IKZF1 and childhood ALL. Several factors should be considered to explain a discrepancy between our results and the previous studies, which include different genotype frequencies in polymorphisms and varied susceptibility to ALL in different ethnic groups. Further studies incorporating larger number of cases and analyzing other possible genes are warranted in childhood ALL. Disclosures:No relevant conflicts of interest to declare.

Mutations of the SF3B1 splicing Factor in Chronic Lymphocytic Leukemia: Association with Progression and Fludarabine-Refractoriness

Abstract 464Fludarabine-refractoriness of chronic lymphocytic leukemia (CLL) is due to TP53 disruption in ∼40% of refractory cases, but in a sizeable fraction of patients the molecular basis of this aggressive clinical phenotype remains unclear. Our initial findings from whole exome sequencing of fludarabine-refractory CLL led to the identification of recurrent mutations of SF3B1, a critical component of the cell spliceosome, prompting further investigations of these alterations in a large CLL panel. The study population comprised 3 clinical cohorts representative of: i) fludarabine-refractory CLL (n=59), including cases (n=11) subjected to whole exome sequencing; ii) newly diagnosed and previously untreated CLL (n=301); and iii) clonally related RS (n=33). Tumor samples were obtained: i) for fludarabine-refractory CLL, immediately before starting the treatment to which the patient eventually failed to respond; ii) for newly diagnosed and previously untreated CLL, at disease presentation. All RS studies were performed on RS diagnostic biopsies. Mutation analysis of SF3B1 was performed on genomic DNA by a combination of Sanger sequencing and targeted next generation sequencing. SF3B1 was altered in 10/59 (17%) fludarabine-refractory CLL by missense mutations (n=9) or in-frame deletions (n=1) clustering in the HEAT3, HEAT4 and HEAT5 repeats of the SF3B1 protein. Two sites that are highly conserved inter-species (codon 662 and codon 700) were recurrently mutated in 3 and 5 cases, respectively. SF3B1 mutations were monoallelic, and were predicted to be functionally significant according to the PolyPhen-2 algorithm. Mutations occurred irrespective of IGHV mutation status, CD38 expression and ZAP70 expression. At the time of fludarabine-refractoriness, SF3B1 mutations were enriched in cases harboring a normal FISH karyotype (p=.008) and distributed in a mutually exclusive fashion with TP53 disruption (mutual information I =0.0609; p=.046). By combining SF3B1 mutations with other genetic lesions enriched in chemorefractory cases (TP53 disruption, NOTCH1 mutations, ATM deletion), fludarabine-refractory CLL appeared to be characterized by multiple molecular alterations that, to some extent, are mutually exclusive. We then compared the prevalence of mutations observed at the time of fludarabine-refractoriness to that observed in other disease phases. At diagnosis, SF3B1 mutations were rare (17/301; 5%), and showed a crude association with short treatment free survival (p<.001) and overall survival (p=.011). Remarkably, 5/17 (29%) CLL mutated at diagnosis were primary fludarabine-refractory patients. In CLL investigated at diagnosis, the hotspot distribution and molecular spectrum of SF3B1 mutations, as well as their mutual relationship with other genetic lesions, were similar to those observed in fludarabine-refractory CLL. SF3B1 mutations were restricted to 2/33 (6.0%) clonally-related RS. Across the different disease phases investigated, mutations were somatically acquired in all cases (n=18) for which germline DNA was available. These data document that mutations of SF3B1, a splicing factor that is a critical component of the spliceosome; i) recurrently associate with fludarabine-refractory CLL; ii) occur at a low rate at CLL presentation; iii) play a minor role in RS transformation, corroborating the notion that CLL histologic shift is molecularly distinct from chemorefractory progression without RS transformation. The identification of SF3B1 mutations points to the involvement of splicing regulation as a novel pathogenetic mechanism in CLL. The pathogenicity of SF3B1 mutations in CLL is strongly supported by clustering of these mutations in evolutionarily conserved hotspots localized within HEAT domains, which are tandemly arranged curlicue-like structures serving as flexible scaffolding on which other components can assemble. Also, the observation that SF3B1 regulates the alternative splicing program of genes controlling cell cycle progression and apoptosis points to a potential contribution of SF3B1 mutations in modulating tumor cell proliferation and survival. In addition to pathogenetic implications, SF3B1 mutations might also provide a therapeutic target for SF3B1 inhibitors, that are currently under pre-clinical development as anti-cancer drugs. Disclosures:No relevant conflicts of interest to declare.

Evidence that polymorphisms in detoxification genes modulate the susceptibility for sporadic medullary thyroid carcinoma

Polymorphic low-penetrance genes have been consistently associated with the susceptibility to a series of human tumors, including differentiated thyroid cancer. To determine their role in medullary thyroid cancer (MTC), we used TaqMan SNP method to genotype 47 sporadic MTC (s-MTC) and a control group of 578 healthy individuals for CYP1A2*F, CYP1A1m1, GSTP1, NAT2 and 72TP53. A logistic regression analysis showed that NAT2C/C (OR=3.87; 95% CI=2.11-7.10; P=2.2×10(-5)) and TP53C/C genotypes (OR=3.87; 95% CI=1.78-6.10; P=2.8×10(-4)) inheritance increased the risk of s-MTC. A stepwise regression analysis indicated that TP53C/C genotype contributes with 8.07% of the s-MTC risk. We were unable to identify any relationship between NAT2 and TP53 polymorphisms suggesting they are independent factors of risk to s-MTC. In addition, there was no association between the investigated genes and clinical or pathological features of aggressiveness of the tumors or the outcome of MTC patients. In conclusion, we demonstrated that detoxification genes and apoptotic and cell cycle control genes are involved in the susceptibility of s-MTC and may modulate the susceptibility to the disease.

Haplotypes of DNA repair and cell cycle control genes, X-ray exposure, and risk of childhood acute lymphoblastic leukemia

BackgroundAcute leukemias of childhood are a heterogeneous group of malignancies characterized by cytogenetic abnormalities, such as translocations and changes in ploidy. These abnormalities may be influenced by altered DNA repair and cell cycle control processes.MethodsWe examined the association between childhood acute lymphoblastic leukemia (ALL) and 32 genes in DNA repair and cell cycle pathways using a haplotype-based approach, among 377 childhood ALL cases and 448 controls enrolled during 1995–2002.ResultsWe found that haplotypes in APEX1, BRCA2, ERCC2, and RAD51 were significantly associated with total ALL, while haplotypes in NBN and XRCC4, and CDKN2A were associated with structural and numerical change subtypes, respectively. In addition, we observed statistically significant interaction between exposure to 3 or more diagnostic X-rays and haplotypes of XRCC4 on risk of structural abnormality-positive childhood ALL.ConclusionsThese results support a role of altered DNA repair and cell cycle processes in the risk of childhood ALL, and show that this genetic susceptibility can differ by cytogenetic subtype and may be modified by exposure to ionizing radiation. To our knowledge, our study is the first to broadly examine the DNA repair and cell cycle pathways using a haplotype approach in conjunction with X-ray exposures in childhood ALL risk. If confirmed, future studies are needed to identify specific functional SNPs in the regions of interest identified in this analysis.Electronic supplementary materialThe online version of this article (doi:10.1007/s10552-011-9848-y) contains supplementary material, which is available to authorized users.

Replicate and die for your own good: Endoreduplication and cell death in the cereal endosperm

Development of the cereal endosperm involves different stages that include cell proliferation, endoreduplication, the biosynthesis and accumulation of storage compounds, and programmed cell death (PCD). During endoreduplication, repeated genome duplications without mitosis or cytokinesis result in large polyploidy nuclei. Endoreduplication is affected by genetic, epigenetic, hormonal and environmental factors, as well as by several cell cycle-controlling genes. Although endoreduplication is strongly correlated with an increase in cell size, the accumulation of storage reserves, and high metabolic rates, its exact role(s) in endosperm development has not been demonstrated.Two distinct PCD types take place in the endosperm. In the starchy endosperm, cells undergo PCD during the maturation phase of seed development, but their corpses are largely retained until germination. Aleurone cells, instead, remain alive throughout endosperm development, but they die and disassemble as a result of the activation of a specific autolytic program during germination. Hydrolases released by dying aleurone cells help digest and remobilize the storage compounds in the starchy endosperm to support seedling development. Although cell death programs in the starchy endosperm and aleurone are characterized by different mechanisms and regulation, the balance between cell death-promoting and -repressing hormones plays a key role in both systems.

Subchronic Pulmonary Pathology, Iron Overload, and Transcriptional Activity after Libby Amphibole Exposure in Rat Models of Cardiovascular Disease

Background: Surface-available iron (Fe) is proposed to contribute to asbestos-induced toxicity through the production of reactive oxygen species.Objective: Our goal was to evaluate the hypothesis that rat models of cardiovascular disease with coexistent Fe overload would be increasingly sensitive to Libby amphibole (LA)-induced subchronic lung injury.Methods: Male healthy Wistar Kyoto (WKY), spontaneously hypertensive (SH), and SH heart failure (SHHF) rats were intratracheally instilled with 0.0, 0.25, or 1.0 mg LA (with saline as the vehicle). We examined bronchoalveolar lavage fluid (BALF) and histological lung sections after 1 week, 1 month, or 3 months for pulmonary biomarkers and pathology. SHHF rats were also assessed at 6 months for pathological changes.Results: All animals developed concentration- and time-dependent interstitial fibrosis. Time-dependent Fe accumulation occurred in LA-laden macrophages in all strains but was exacerbated in SHHF rats. LA-exposed SHHF rats developed atypical hyperplastic lesions of bronchiolar epithelial cell origin at 3 and 6 months. Strain-related baseline differences existed in gene expression at 3 months, with persistent LA effects in WKY but not SH or SHHF rats. LA exposure altered genes for a number of pathways, including inflammation, immune regulation, and cell-cycle control. Cell-cycle control genes were inhibited after LA exposure in SH and SHHF but not WKY rats, whereas tumor suppressor genes were induced only in WKY rats. The inflammatory gene expression also was apparent only in WKY rats.Conclusion: These data show that in Fe-overload conditions, progressive Fe accumulation occurs in fiber-laden macrophages within LA-induced lesions. Fe overload does not appear to contribute to chronic inflammation, and its role in hyperplastic lesion development requires further examination.

The E-box Binding Factors Max/Mnt, MITF, and USF1 Act Coordinately with FoxO to Regulate Expression of Proapoptotic and Cell Cycle Control Genes by Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3 Signaling

Phosphatidylinositol (PI) 3-kinase/Akt signaling plays a critical role in cell proliferation and survival, partly by regulation of FoxO transcription factors. Previous work using global expression profiling indicated that inhibition of PI 3-kinase in proliferating cells led to induction of genes that promote cell cycle arrest and apoptosis. The upstream regulatory regions of these genes had binding sites not only for FoxO, but also for Myc/Max transcription factors. In the present study, we have addressed the role of Myc family members and related E-box-binding proteins in the regulation of these genes. Chromatin immunoprecipitations and RNA interference indicated that transcription was repressed by Max-Mnt-Sin3a-histone deacetylase complexes in proliferating cells. Inhibition of PI 3-kinase led to a loss of Max/Mnt binding and transcriptional induction by MITF and USF1, as well as FoxO. Both MITF and USF1 were activated by glycogen synthase kinase (GSK) 3, with GSK3 phosphorylation sites on USF1 identified as the previously described activating site threonine 153 as well as serine 186. siRNA against MITF as well as against FoxO3a protected cells from apoptosis following PI 3-kinase inhibition. These results define a novel E-box-regulated network that functions coordinately with FoxO to regulate transcription of apoptotic and cell cycle regulatory genes downstream of PI 3-kinase/Akt/GSK3 signaling.

Aberrant methylation in promoter-associated CpG islands of multiple genes in chronic myelogenous leukemia blast crisis

Chronic myelogenous leukemia (CML) has a typical progressive course with transition from a chronic phase to a terminal blast crisis phase. The mechanisms that lead to disease progression remain to be elucidated. To understand the role of aberrant methylation in the progression of CML, DNA methylation patterns in 16 patients with CML blast crisis were analyzed. Methylation status was analyzed by methylation-specific PCR (MSP) for 13 genes, including cell cycle regulating genes, DNA repair genes, apoptosis-related genes, a differentiation-associated gene and a cytokine signaling gene. The frequency of samples with methylation in each of the following genes were: p15, 18%; MGMT, 12%; RARβ, 12%; p16, 6%; DAPK, 6% and FHIT, 6%. In total, four (25%) cases showed methylation of at least one gene. None of the 16 cases showed hypermethylation of the hMLH1 or hMSH2 genes. These results suggest that hypermethylation of cell cycle control genes, but not DNA mismatch repair genes, play a significant role in the progression of CML.

Deregulation of Cancer-Related Pathways in Primary Hepatocytes Derived from DNA Repair-Deficient Xpa−/−p53+/− Mice upon Exposure to Benzo[a]pyrene

The current method to predict carcinogenicity of chemicals or drugs is the chronic 2-year rodent bioassay, which has disadvantages in duration, animal use, and specificity. An attractive alternative is the DNA repair-deficient Xpa(-/-)p53(+/-) mouse model that is sensitive to both genotoxic and nongenotoxic carcinogens. A next step in alternative carcinogenicity testing is the development of reliable in vitro systems. We investigated the use of primary hepatocytes, isolated from wild-type (WT) and Xpa(-/-)p53(+/-) mice, in combination with transcriptome analyses for their usefulness to predict carcinogenic features of compounds. As a proof of principle, we studied the response of hepatocytes to the genotoxic carcinogen benzo[a]pyrene (B[a]P). Upon treatment, both WT and Xpa(-/-)p53(+/-) hepatocytes appeared to be metabolically active. However, Xpa(-/-)p53(+/-) hepatocytes were more sensitive than WT hepatocytes to B[a]P treatment in terms of cell survival. In B[a]P-treated WT hepatocytes, DNA repair and cell cycle control genes were transcriptionally activated. Xpa(-/-)p53(+/-) hepatocytes were more responsive to B[a]P exposure, resulting in the downregulation of cancer-related pathways. Deregulation of mitogen-activated protein kinase signaling seems to play an essential role in this and might be the underlying reason for the increased susceptibility of Xpa(-/-)p53(+/-) mice toward carcinogens. Our conclusion is that primary hepatocytes combined with transcriptomics are promising to identify the carcinogenic features of chemicals. Furthermore, these cells seem suitable to gain further insight into the molecular mechanisms of the increased sensitivity of Xpa(-/-)p53(+/-) mice toward both genotoxic and nongenotoxic carcinogens.

Right‐side and left‐side colon cancer follow different pathways to relapse

There is growing evidence that cancer of the ascending (right-side) colon is different from cancer of the descending (left-side) colon at the molecular level. Using microarray data from 102 right-side colon carcinomas and 95 left-side colon carcinomas we show that different pathways dominate progression to relapse in right-side and left-side colon cancer. Right-side tumors at a high risk for relapse exhibit elevated expression of cell cycle control genes and elevated Wnt signaling. On the other hand, relapse-prone left-side tumors show elevated expression of genes that promote stromal expansion and reduced expression of tumor suppressor genes that initiate Wnt signaling. Single gene prognostic biomarkers are found separately for right-side and left-side disease. In left-side tumors with low expression levels of NADPH oxidase 4 (NOX4) the 5-yr relapse-free survival probability is 0.89 95% CI (0.80-0.99), and in tumors with elevated NOX4 expression the probability is 0.51 95% CI (0.37-0.70). Right-side tumors with elevated expression levels of caudal type homeobox 2 (CDX2) have a 5-yr relapse-free survival probability of 0.88 95% CI (0.80-0.96), and those with low CDX2 expression have a corresponding probability of 0.39 95% CI (0.15-0.78). Both NOX4 and CDX2 are much less prognostic on the opposite sides. This newly identified role of NOX4 in colon cancer is further investigated using the SW620 lymph node metastasis colon adenocarcinoma cell line and RNA interference. We show that NOX4 is expressed in the SW620 cell line and that application of NOX4 siRNA causes a significant reduction in reactive oxidative species production.