Genes Controlling Cell Proliferation Research Articles

Cell Type-Dependent Function of Atf5: Where We Go from Here

Copyright: © 2012 Lengel KL, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Uncontrollable cell proliferation and suppressed apoptosis are hallmarks of tumorigenic transformation [1-3]. Deregulation of genes controlling cell proliferation and survival plays an important role in the process. For instance, apoptotic genes are frequently down-regulated while anti-apoptotic genes are highly expressed in a number of cancer cell types; artificial down regulation of anti-apoptotic genes or up regulation of apoptotic genes are often sufficient to eradicate those cancer cells. The activating transcription factor 5 (ATF5 or ATFx) is a member of the ATF/CREB transcription factor family. Although ATF5 is known to regulate cell cycle progression [4-7], cell survival [5-13], autophagy [14], cell fate determination [15-17] and cellular stress response [13-19], and it is likely involved in the development of schizophrenia [20-22] and chronic lymphocytic leukemia [23], only a few of its targets have been reported and the mechanism of ATF5 function remains largely unknown and occasionally controversial.

Recent advances in understanding the role of diet and obesity in the development of colorectal cancer

Colorectal cancer (CRC) is a major cause of premature death in the UK and many developed countries. However, the risk of developing CRC is well recognised to be associated not only with diet but also with obesity and lack of exercise. While epidemiological evidence shows an association with factors such as high red meat intake and low intake of vegetables, fibre and fish, the mechanisms underlying these effects are only now being elucidated. CRC develops over many years and is typically characterised by an accumulation of mutations, which may arise as a consequence of inherited polymorphisms in key genes, but more commonly as a result of spontaneously arising mutations affecting genes controlling cell proliferation, differentiation, apoptosis and DNA repair. Epigenetic changes are observed throughout the progress from normal morphology through formation of adenoma, and the subsequent development of carcinoma. The reasons why this accumulation of loss of homoeostatic controls arises are unclear but chronic inflammation has been proposed to play a central role. Obesity is associated with increased plasma levels of chemokines and adipokines characteristic of chronic systemic inflammation, and dietary factors such as fish oils and phytochemicals have been shown to have anti-inflammatory properties as well as modulating established risk factors such as apoptosis and cell proliferation. There is also some evidence that diet can modify epigenetic changes. This paper briefly reviews the current state of knowledge in relation to CRC development and considers evidence for potential mechanisms by which diet may modify risk.

Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

MYC regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis. MYC also controls the expression of DNA double-strand break (DSB) repair genes and therefore may be a potential target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability. In this report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survival in response to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associates with several DSB repair gene promoters including Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2, DNA-PKcs, XRCC4, Ku70, and DNA ligase IV. Endogenous MYC protein expression was associated with increased RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induction of MYC in G(0)-G(1) and S-G(2)-M cells resulted in upregulation of Rad51 gene expression. MYC knockdown using small interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologous recombination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYC resulted in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However, MYC-dependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycin C or ionizing radiation, two agents selectively toxic to DSB repair-deficient cells. Our results suggest that anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensitization or chemosensitization.

Abstract B13: R-spondin3 overexpression induces transformation and deregulates expression of genes controlling cell proliferation and apoptosis in NIH3T3 cells

Abstract It has been reported that R-spondin3 (Rspo3) is a common insertion site for the viral genome in mouse mammary tumor virus (MMTV) induced-mammary tumors and that HC11 cells over-expressing this protein become tumorigenic when transplanted into mice. In addition, we have found that Rspo-3 over-expression is associated with loss of hormone regulation of initially pregnancy-dependent MMTV-induced mouse mammary tumors. In order to determine whether this gene may behave as an oncogen in culture, even in cells that do not derive from mammary tissue, NIH3T3 cells were stably transfected with mouse Rspo3 full-length cDNA under the control of the cytomegalovirus (CMV) promoter (3T3-Rspo3 cells). We intend this cell line to provide a tool that might allow the study of signaling pathways altered by Rspo-3 expression We found that 3T3-Rspo3 cells showed the capacity to form foci when cultured on top of a lawn of normal NIH3T3 cells attached to plastic dishes and to grow as colonies when seeded in soft-agar. To determine signaling transduction pathways that might be altered by Rspo3 over-expression, we analyzed the level of expression of phosphorylated proteins critical in the regulation of cell growth or death and found that transfected cells showed higher levels of phosphorylated AKT and p38 MAPK, while no significant differences were found when the levels of other phosphorylated MAPKs (as Erk1/2) were analyzed. Besides, crystal violet staining assays revealed that Rspo3 transfected cells were less dependent on serum concentration to grow in culture. Interestingly, survival of 3T3-Rspo3 cells was reduced by treatment with a potent and selective phosphatidylinositol-3 kinase (PI-3K) pharmacological inhibitor, LY # 294002, a well-known regulator of signaling by AKT. In addition, expression of Cyclin D1, a protein associated with cell proliferation, was significantly increased and correlated with Rspo3 overexpression. To determine other genes that might be involved in 3T3-Rspo3 cell survival, we studied the level of expression of Bcl family members. We found that the cells over-expressing Rspo3 showed diminished expression of pro-apoptotic proteins BAD and BAX, comparing to control cells. On the other hand, we observed higher expression of the anti-apoptotic (long) Bcl-X form. Besides, preliminary results indicate that LY # 294002 treatments alter the expression pattern of Bcl family members at the protein level, suggesting that AKT phosphorylation might be involved in regulation of the balance between pro- and anti-apoptotic proteins. In summary, we have developed a tool that can be used to dissect signaling pathways that may be responsible for the transforming effects triggered by over-expression of Rspo3. Our results, added to the reported in vivo studies, provide evidences that Rspo3 can be fully characterized as an oncogene. In addition, we have determined that activation of the PI-3K/AKT pathway might be involved in such an activity. Citation Information: Cancer Res 2009;69(23 Suppl):B13.

Quantitative Proteomics Reveals Regulation of Dynamic Components within TATA-binding Protein (TBP) Transcription Complexes

Affinity purification in combination with isotope labeling of proteins has proven to be a powerful method to discriminate specific from nonspecific interactors. However, in the standard SILAC (stable isotope labeling by amino acids in cell culture) approach dynamic components may easily be assigned as nonspecific. We compared two affinity purification protocols, which in combination revealed information on the dynamics of protein complexes. We focused on the central component in eukaryotic transcription, the human TATA-binding protein, which is involved in different complexes. All known TATA-binding protein-associated factors (TAFs) were detected as specific interactors. Interestingly one of them, BTAF1, exchanged significantly in cell extracts during the affinity purification. The other TAFs did not display this behavior. Cell cycle synchronization showed that BTAF1 exchange was regulated during mitosis. The combination of the two affinity purification protocols allows a quantitative approach to identify transient components in any protein complex.

Retinoblastoma Protein and the Leukemia-Associated PLZF Transcription Factor Interact To Repress Target Gene Promoters.

Translocations of the retinoic acid receptor alpha (RARA) locus with the PLZF or PML genes lead to expression of oncogenic PLZF-RARα or PML-RARα fusion proteins, respectively. These fusion oncoproteins constitutively repress RARα target genes, in large part through aberrant recruitment of multiprotein co-repressor complexes. PML and PML-RARα have previously been shown to associate with the retinoblastoma (Rb) tumour suppressor protein in its hypophosphorylated state. Here we demonstrate that PLZF also interacts with Rb in vitro and in vivo. The interaction between PLZF and Rb is mediated through the Rb pocket and the region of PLZF that lies between its transcriptional repression (POZ) and DNA binding (zinc-finger) domains. Additionally, Rb can simultaneously interact with PLZF and the E2F1 S phase-inducing transcription factor, suggesting that these proteins can exist in the same multiprotein complex. In contrast to the interaction between PML or E2F1 with Rb, the PLZF-Rb interaction is not dependent on hypophosphorylation of Rb. The interaction between PLZF and Rb is further underlined by chromatin immunoprecipitation analysis of PLZF binding to genomic DNA, which shows that PLZF associates with genes controlling cell proliferation known to be regulated by Rb and E2F (for example cdc6). Co-expression of PLZF and Rb results in enhancement of transcriptional repression of PLZF and E2F target genes, indicating functional co-operation between the two proteins. Both PLZF and Rb have been shown to have roles in stem cell biology and, taken together, these data provide a plausible scenario in which interactions between PLZF and Rb function in stem cell commitment or maintenance and self-renewal. The oncogenic PLZF-RARα fusion also interacts with Rb, suggesting that deregulation of Rb function may be a factor in the molecular pathogenesis of PLZF-RARα associated acute promyelocytic leukemia.

Aβ treatment and P301L tau expression in an Alzheimer's disease tissue culture model act synergistically to promote aberrant cell cycle re‐entry

Microarrays enable the observation of gene expression in experimental models of Alzheimer's disease (AD), with implications for the human pathology. Histopathologically, AD is characterized by Abeta-containing plaques and tau-containing neurofibrillary tangles. Here, we used a human SH-SY5Y neuroblastoma cell system to assess the role of P301L mutant human tau expression, and treatment with or without Abeta on gene regulation. We found that Abeta and P301L tau expression independently affect the regulation of genes controlling cell proliferation and synaptic elements. Moreover, Abeta and P301L tau act synergistically on cell cycle and DNA damage genes, yet influence specific genes within these categories. By using neuronally differentiated P301L tau cells, we can show that Abeta treatment induces an early upregulation of cell cycle control and synaptic genes. At the protein level, by using Kinetworks multi-immunoblotting and BrdU labelling, we found that although P301L tau and Abeta both affected levels of cell cycle proteins, their effects were distinct, in particular concerning DNA damage proteins. Moreover, DNA synthesis was observed only when SH-SY5Y cells overexpressed human wild-type or P301L tau and were incubated with Abeta. Thus, our study shows that Abeta treatment and human tau overexpression in an AD cell culture model act synergistically to promote aberrant cell cycle re-entry, supporting the mitosis failure hypothesis in AD.

RNA interference technologies and their use in cancer research

Recently, RNA interference has evolved into a powerful research tool to functionally characterize genes. Genome-wide RNA interference reagents can study the loss-of-function phenotypes of candidate genes in the context of various disease model systems. In this review, we discuss the data from the most recent studies using RNA interference reagents with a focus on RNA interference-based genomic screening as a tool to expand our knowledge about the molecular basis of cancer. Tumorigenesis is the result of the progressive accumulation of mutations in genes controlling cell proliferation and death. Various genes carrying these alterations are known to be directly linked to tumor growth; however, how to translate this knowledge into effective chemotherapeutics, nontoxic to normal cells, is still a subject of intensive research. Loss-of-function studies offer a potential for validation of known and unrecognized tumor-associated targets. RNA interference-mediated gene knockdown can be exploited to study the reprogrammed circuitry of genes, discover gene interactions restricted to cancer cells and identify mechanisms of chemoresistance in cancer cells. In addition, the simultaneous use of cancer drugs and RNA interference also provides a paradigm to develop strategies to inactivate essential genes promoting neoplastic growth.

Sinusoidal swinging dynamics of the telomere repair and cell growth activation functions of telomerase in rat liver cancer cells

Telomerase is a multimolecular complex of reverse transcriptase, RNA template, and regulatory proteins. It has two known functions: catalysis of the addition of [TTAGGG] repeats to telomeric DNA and the activation of various genes controlling cell proliferation. The possible coordination of these two functions is a key issue in understanding the growth of cancer cells. We report long-term changes to this complex system, as shown by specific data analysis methods. We show that the dynamics of the two functions of telomerase are tightly linked, with a change in predominant function every 13–14 weeks. The conservative behavior of this dynamic system probably accounts for the persistent proliferation of cancer cells.

Transcriptional profile of mouse pre-granulosa and Sertoli cells isolated from early-differentiated fetal gonads

Gonadal sex determining (GSD) genes that initiate fetal ovarian and testicular development and differentiation are expressed in the cells of the urogenital ridge that differentiate as somatic support cells (SSCs), i.e., granulosa cells of the ovary and Sertoli cells of the testis. To identify potential new mammalian GSD genes, we used the Mouse Genome 430v2.0 GeneChip™ to analyze gene expression differences between XX and XY SSCs cells isolated from the gonads of embryonic day (E) 13 C57BL/6J fetuses carrying an EGFP reporter transgene expressed specifically in SSCs. In addition, genome wide expression differences between XX and XY E13 whole gonads were examined. Analysis revealed that XX and XY E13 SSCs differentially express 647 transcripts (False Discovery Rate cutoff ⩽1%), including transcripts not previously reported to exhibit a sexually dimorphic expression pattern in this unique cell population. Enrichment for genes controlling cell proliferation was noted in XY SSCs, whereas enrichment for genes controlling cell morphology and metabolic status was identified in XX SSCs. Among the newly identified differentially expressed transcripts are potential GSD genes involved in unexplained human sex reversal cases.

Medakasimplet(FAM53B) belongs to a family of novel vertebrate genes controlling cell proliferation

The identification of genes that regulate proliferation is of great importance to developmental biology, regenerative medicine and cancer research. Using an in situ screen on a cortical structure of the medaka fish brain, we identified the simplet gene (smp), which is homologous to the human FAM53B gene. smp was expressed in actively proliferating cells of the CNS throughout embryogenesis. It belongs to a family of vertebrate-specific genes with no characterized biochemical domains. We showed that FAM53B bound 14-3-3 chaperones, as well as SKIIP proteins, adaptor proteins connecting DNA-binding proteins to modulators of transcription. smp inactivation with morpholinos led to delayed epiboly and reduced embryonic size. Absence of Smp activity did not induce apoptosis, but resulted in a reduced cell proliferation rate and enlarged blastomeres. Moreover, smp was shown to control the expression of the pluripotency-associated oct4/pou5f1 gene. We propose that smp is a novel vertebrate-specific gene needed for cell proliferation and that it is probably associated with the maintenance of a pluripotent state.

The Growth Regulators warts/ lats and melted Interact in a Bistable Loop to Specify Opposite Fates in Drosophila R8 Photoreceptors

Color vision in Drosophila relies on the comparison between two color-sensitive photoreceptors, R7 and R8. Two types of ommatidia in which R7 and R8 contain different rhodopsins are distributed stochastically in the retina and appear to discriminate short (p-subset) or long wavelengths (y-subset). The choice between p and y fates is made in R7, which then instructs R8 to follow the corresponding fate, thus leading to a tight coupling between rhodopsins expressed in R7 and R8. Here, we show that warts, encoding large tumor suppressor (Lats) and melted encoding a PH-domain protein, play opposite roles in defining the yR 8 or pR8 fates. By interacting antagonistically at the transcriptional level, they form a bistable loop that insures a robust commitment of R8 to a single fate, without allowing ambiguity. This represents an unexpected postmitotic role for genes controlling cell proliferation (warts and its partner hippo and salvador) and cell growth (melted).

TAF4 inactivation in embryonic fibroblasts activates TGF beta signalling and autocrine growth.

We have inactivated transcription factor TFIID subunit TBP-associated factor 4 (TAF4) in mouse embryonic fibroblasts. Mutant taf4(-/-) cells are viable and contain intact TFIID comprising the related TAF4b showing that TAF4 is not an essential protein. TAF4 inactivation deregulates more than 1000 genes indicating that TFIID complexes containing TAF4 and TAF4b have distinct target gene specificities. However, taf4(-/-) cell lines have altered morphology and exhibit serum-independent autocrine growth correlated with the induced expression of several secreted mitotic factors and activators of the transforming growth factor beta signalling pathway. In addition to TAF4 inactivation, many of these genes can also be induced by overexpression of TAF4b. A competitive equilibrium between TAF4 and TAF4b therefore regulates expression of genes controlling cell proliferation. We have further identified a set of genes that are regulated both by TAF4 and upon adaptation to serum starvation and which may be important downstream mediators of serum-independent growth. Our study also shows that TAF4 is an essential cofactor for activation by the retinoic acid receptor and CREB, but not for Sp1 and the vitamin D3 receptor.

Developmental instability of the Drosophila wing as an index of genomic perturbation and altered cell proliferation

We experimentally induced different levels of instability affecting the development of specific wing regions of Drosophila melanogaster using the UAS-GAL4 system. A common index of developmental instability is fluctuating asymmetry (FA), that is, random differences between body sides of single individuals. We studied the FA in transgenic strains carrying random genomic insertions (UAS strains), as well as insertions in the regulatory region of genes involved in the organization of wing development (GAL4 strains). In addition, the expression of genes that increase (dp110 and 3622) or decrease (dPTEN) cell proliferation was ectopically induced. Our results are related to different levels of perturbation. Through the first kind of perturbation, genome integrity was compromised by the insertion of foreign DNA. In all cases, we observed a general increase in FA, although it was rarely found significant. The second kind of perturbation involved a modification of genes controlling wing development through the insertion of a GAL4 sequence in their promoter region. The third kind involved the ectopic expression of genes controlling cell proliferation. Our results show that (i) the level of FA is connected with the level of morphological perturbation induced, (ii) FA increase was higher in the wing regions that were the target of the genetic perturbation, and (iii) developmental instability was also observed in regions that were not directly addressed by the perturbation. The results were discussed on the basis of the running models about Drosophila wing development.

Gene expression profiling identifies activating transcription factor 3 as a novel contributor to the proapoptotic effect of curcumin

The antitumor effect of curcumin (diferuloylmethane) is well established. However, there have been no unbiased studies to identify novel molecular targets of this compound. We therefore undertook a gene expression profiling study to identify novel targets of curcumin. A cDNA array comprised of 12,625 probes was used to compare total RNA extracted from curcumin-treated and untreated MDA-1986 cells for differential gene expression. We identified 202 up-regulated mRNAs and 505 transcripts decreased > or =2-fold. The proapoptotic activating transcription factor 3 (ATF3) was induced >4-fold. Two negative regulators of growth control [antagonizer of myc transcriptional activity (Mad) and p27kip1] were induced 68- and 3-fold, respectively. Additionally, two dual-activity phosphatases (CL 100 and MKP-5), which inactivate the c-jun-NH2-kinases, showed augmented expression, coinciding with reduced expression of the upstream activators of c-jun-NH2-kinase (MEKK and MKK4). Of the repressed genes, the expression of Frizzled-1 (Wnt receptor) was most strongly attenuated (8-fold). Additionally, two genes implicated in growth control (K-sam, encoding the keratinocyte growth factor receptor, and HER3) as well as the E2F-5 transcription factor, which regulates genes controlling cell proliferation, also showed down-regulated expression. Considering its role in apoptosis, we determined the contribution of ATF3 to the antitumor effect of curcumin. Curcumin-treated MDA-1986 cells showed a rapid, dose-dependent increase in ATF3/mRNA protein. Moreover, expression of an exogenous ATF3 cDNA synergized with curcumin in inducing apoptosis. Thus, we have identified several putative, novel molecular targets of curcumin and showed that one, (ATF3) contributes to the proapoptotic effects of this compound.

Dysplasia in inflammatory bowel diseases: definition and clinical impact.

Dysplasia is a morphological term that ethymologically means 'malformation'. For the definition of inflammatory bowel disease-related dysplasia, the nature and origin of the malformation are stressed and the lesion is defined as an epithelial malformation that is unequivocally neoplastic but noninvasive. The use of a precise definition is necessary because of the clinical consequences related to the finding of dysplasia in IBD. The microscopic diagnosis of dysplasia, however, remains difficult. Clinically, it is important to make a proper differential diagnosis between polypoid IBD-related dysplasia and sporadic adenoma occurring in IBD, and between therapy-related 'pseudodysplasia' and genuine dysplasia. When dysplasia is diagnosed, a second opinion may be indicated because of the clinical consequences. Additional techniques to search for genetic defects associated with carcinogenesis can help to support the diagnosis. They can identify changes in DNA content and molecular changes resulting from defects of genes controlling cell proliferation and death or tissue structure. These changes can, however, be absent, appear early or late in the transition from normality toward dysplasia and cancer, or appear during repair. Positive findings indicate an increased cancer risk, but the magnitude of the risk remains to be defined. A positive diagnosis of genuine dysplasia necessitates clinical action - either follow-up of the patient or treatment. In practice, treatment means surgery because dysplasia can be a precursor and/or a marker of malignancy, except for sporadic adenomas, which can be removed locally.

Role of Transforming Growth Factor β1 (TGF β1) in Mediating Androgen-induced Growth Inhibition in Human Adrenal Cortex in Vitro

We have previously found that the androgen receptor gene is expressed both in normal and adenomatous human adrenal cortex and in the NCI-H295 human adrenocortical cancer cell line. Furthermore, we have observed that dihydrotestosterone (DHT) at physiological concentrations (10 −11 M) inhibits human adrenocortical cell growth in vitro and slightly decreases c-myc RNA levels in NCI-H295 cells. As c-myc is probably not the main mechanism mediating DHT-induced inhibition of cell growth, other genes controlling cell proliferation may be involved. Transforming Growth Factor beta (TGF β) is a regulatory peptide that acts by both autocrine and paracrine mechanisms to control proliferation and differentiation, and there is previous evidence that TGF β may exert an antimitotic effect on human fetal adrenal cells in vitro. This study examines a possible role for TGF β 1 in mediating the DHT-induced reduction of human adrenocortical cell growth. TGF β1 and its receptor (TGF β RII) are expressed in DHT-treated and nontreated NCI-H295 cells; on Northern blot analysis 24-h treatment with DHT (10 −11 M) produced a small increase in TGF β RII RNA, and quantitative RT-PCR showed a 1.5-fold increase in TGF β 1 RNA levels. These findings suggest that TGF β 1 and its receptor may be involved in DHT-induced inhibition of human adrenocortical cell growth.