E2F4-RB and E2F4-p107 complexes suppress gene expression by transforming growth factor β through E2F binding sites

Transforming growth factor β (TGF-β) causes growth arrest in most cell types. TGF-β induces hypophosphorylation of retinoblastoma susceptibility gene 1 product (RB), which sequesters E2F factors needed for progression into S phase of the cell cycle, thereby leading to cell cycle arrest at G1. It is possible, however, that the E2F-RB complex induced by TGF-β may bind to E2F sites and suppress expression of specific genes whose promoters contain E2F binding sites. We show here that TGF-β treatment of HaCaT cells induced the formation of E2F4-RB and E2F4-p107 complexes, which are capable of binding to E2F sites. Disruption of their binding to DNA with mutation in the E2F sites did not change the expression from promoters of E2F1, B-myb, or HsORC1 genes in cycling HaCaT cells. However, the same mutation stimulated 5- to 6-fold higher expression from all three promoters in cells treated with TGF-β. These results suggest that E2F binding sites play an essential role in the transcription repression of these genes under TGF-β treatment. Consistent with their repression of TGF-β-induced gene expression, introduction of E2F sites into the promoter of cyclin-dependent kinase inhibitor p15INK4B gene effectively inhibited its induction by TGF-β. Experiments utilizing Gal4-RB and Gal4-p107 chimeric constructs demonstrated that either RB or p107 could directly repress TGF-β induction of p15INK4B gene when tethered to p15INK4B promoter through Gal4 DNA binding sites. Therefore, E2F functions to bring RB and p107 to E2F sites and represses gene expression by TGF-β. These results define a specific function for E2F4-RB and E2F4-p107 complexes in gene repression under TGF-β treatment, which may constitute an integral part of the TGF-β-induced growth arrest program.

The cdr2+ Gene Encodes a Regulator of G2/M Progression and Cytokinesis in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells respond to nutrient deprivation by altering G2/M cell size control. The G2/M transition is controlled by activation of the cyclin-dependent kinase Cdc2p. Cdc2p activation is regulated both positively and negatively. cdr2+ was identified in a screen for regulators of mitotic control during nutrient deprivation. We have cloned cdr2+ and have found that it encodes a putative serine-threonine protein kinase that is related to Saccharomyces cerevisiae Gin4p and S. pombe Cdr1p/Nim1p. cdr2+ is not essential for viability, but cells lacking cdr2+ are elongated relative to wild-type cells, spending a longer period of time in G2. Because of this property, upon nitrogen deprivation cdr2+ mutants do not arrest in G1, but rather undergo another round of S phase and arrest in G2 from which they are able to enter a state of quiescence. Genetic evidence suggests that cdr2+ acts as a mitotic inducer, functioning through wee1+, and is also important for the completion of cytokinesis at 36°C. Defects in cytokinesis are also generated by the overproduction of Cdr2p, but these defects are independent of wee1+, suggesting that cdr2+ encodes a second activity involved in cytokinesis.

Mechanisms of G2 Arrest in Response to Overexpression of p53

Overexpression of p53 causes G2 arrest, attributable in part to the loss of CDC2 activity. Transcription of cdc2 and cyclin B1, determined using reporter constructs driven by the two promoters, was suppressed in response to the induction of p53. Suppression requires the regions −287 to −123 of the cyclin B1 promoter and −104 to −74 of the cdc2 promoter. p53 did not affect the inhibitory phosphorylations of CDC2 at threonine 14 or tyrosine 15 or the activity of the cyclin-dependent kinase that activates CDC2 by phosphorylating it at threonine 161. Overexpression of p53 may also interfere with the accumulation of CDC2/cyclin B1 in the nucleus, required for cells to enter mitosis. Constitutive expression of cyclin B1, alone or in combination with the constitutively active CDC2 protein T14A Y15F, did not reverse p53-dependent G2 arrest. However, targeting cyclin B1 to the nucleus in cells also expressing CDC2 T14A Y15F did overcome this arrest. It is likely that several distinct pathways contribute to p53-dependent G2 arrest.

Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN-γ target genes

Most of the activities of IFN-γ are the result of STAT1-mediated transcriptional responses. In this study, we show that the BRCA1 tumor suppressor acts in concert with STAT1 to differentially activate transcription of a subset of IFN-γ target genes and mediates growth inhibition by this cytokine. After IFN-γ treatment, induction of the cyclin-dependent kinase inhibitor, p21WAF1, was synergistically activated by BRCA1, whereas the IRF-1 gene was unaffected. Importantly, the differential induction of p21WAF1 was impaired in breast cancer cells homozygous for the mutant BRCA1 5382C allele. Biochemical analysis illustrated that the mechanism of this transcriptional synergy involves interaction between BRCA1 aa 502–802 and the C-terminal transcriptional activation domain of STAT1 including Ser-727 whose phosphorylation is crucial for transcriptional activation. Significantly, STAT1 proteins mutated at Ser-727 bind poorly to BRCA1, reinforcing the importance of Ser-727 in the recruitment of transcriptional coactivators by STAT proteins. These findings reveal a novel mechanism for BRCA1 function in the IFN-γ-dependent tumor surveillance system.

Increased IGF-II protein affects p57kip2 expression in vivo and in vitro: Implications for Beckwith–Wiedemann syndrome

In both human and mouse, the Igf2 gene, localized on chromosomes 11 and 7, respectively, is expressed from the paternally inherited chromosome in the majority of tissues. Insulin-like growth factor-II (IGF-II) plays an important role in embryonic growth, and aberrant IGF2 expression has been documented in several human pathologies, such as Beckwith–Wiedemann syndrome (BWS), and a wide variety of tumors. Human and mouse genetic data strongly implicate another gene, CDKN1C (p57kip2), located in the same imprinted gene cluster on human chromosome II, in BWS. p57KIP2 is a cyclin-dependent kinase inhibitor and is required for normal mouse embryonic development. Mutations in CDKN1C (p57kip2) have been identified in a small proportion of patients with BWS, and removal of the gene from mice by targeted mutagenesis produces a phenotype with elements in common with this overgrowth syndrome. Patients with BWS with biallelic expression of IGF2 or with a CDKN1C (p57kip2) mutation, as well as overlapping phenotypes observed in two types of mutant mice, the p57kip2 knockout and IGF-II-overexpressing mice, strongly suggest that the genes may act in a common pathway of growth control in situations where Igf2 expression is abnormal. Herein, we show that p57kip2 expression is reduced on IGF-II treatment of primary embryo fibroblasts in a dose-dependent manner. In addition, p57kip2 expression is down-regulated in mice with high serum levels of IGF-II. These data suggest that the effects of increased IGF-II in BWS may, in part, be mediated through a decrease in p57kip2 gene expression.

Adhesion to fibronectin stimulates proliferation of wild-type and bcr/abl-transfected murine hematopoietic cells

Cells of most tissues require adhesion to a surface to grow. However, for hematopoietic cells, both stimulation and inhibition of proliferation by adhesion to extracellular matrix components have been described. Furthermore, it has been suggested that progenitor cells from chronic myelogenous leukemia show decreased β1 integrin-mediated adhesion to fibronectin, resulting in increased proliferation and abnormal trafficking. However, we show here that the chronic myelogenous leukemia-specific fusion protein p210bcr/abl stimulates the expression of α5β1 integrins and induces adhesion to fibronectin when expressed in the myeloid cell line 32D. Moreover, proliferation of both p210bcr/abl-transfected 32D (32Dp210) cells and untransfected 32D cells is stimulated by immobilized fibronectin. Cell cycle analysis revealed that nonadherent 32D and 32Dp210 cells are arrested in late G1 or early S phase, whereas the adherent fractions continue cycling. Although both adherent and nonadherent p210bcr/abl-transfected and parental 32D cells express equal amounts of cyclin A, a protein necessary for cell cycle progression at the G1/S boundary, cyclin A complexes immunoprecipitated from 32D cells cultured on immobilized fibronectin were found to be catalytically inactive in nonadherent but not in adherent cells. In addition, as compared with untransfected 32D cells, cyclin A immunoprecipitates from 32Dp210 cells exhibited a greatly elevated kinase activity and remained partially active irrespective of the adhesion status. The lack of cyclin A/cyclin-dependent kinase (CDK) 2 activity in nonadherent 32D cells appeared to result from increased expression and cyclin A complex formation of the CDK inhibitor p27Kip1. Taken together, our results indicate that adhesion stimulates cell cycle progression of hematopoietic cells by down-regulation of p27Kip1, resulting in activation of cyclin A/CDK2 complexes and subsequent transition through the G1/S adhesion checkpoint.

Ubiquitination of RNA polymerase II large subunit signaled by phosphorylation of carboxyl-terminal domain

A sensitive assay using biotinylated ubiquitin revealed extensive ubiquitination of the large subunit of RNA polymerase II during incubations of transcription reactions in vitro. Phosphorylation of the repetitive carboxyl-terminal domain of the large subunit was a signal for ubiquitination. Specific inhibitors of cyclin-dependent kinase (cdk)-type kinases suppress the ubiquitination reaction. These kinases are components of transcription factors and have been shown to phosphorylate the carboxyl-terminal domain. In both regulation of transcription and DNA repair, phosphorylation of the repetitive carboxyl-terminal domain by kinases might signal degradation of the polymerase.

p27 and Rb are on overlapping pathways suppressing tumorigenesis in mice

The commitment of cells to replicate and divide correlates with the activation of cyclin-dependent kinases and the inactivation of Rb, the product of the retinoblastoma tumor suppressor gene. Rb is a target of the cyclin-dependent kinases and, when phosphorylated, is inactivated. Biochemical studies exploring the nature of the relationship between cyclin-dependent kinase inhibitors and Rb have supported the hypothesis that these proteins are on a linear pathway regulating commitment. We have been able to study this relationship by genetic means by examining the phenotype of Rb+/−p27−/− mice. Tumors arise from the intermediate lobe cells of the pituitary gland in p27−/− mice, as well as in Rb+/− mice after loss of the remaining wild-type allele of Rb. Using these mouse models, we examined the genetic interaction between Rb and p27. We found that the development of pituitary tumors in Rb+/− mice correlated with a reduction in p27 mRNA and protein expression. To determine whether the loss of p27 was an indirect consequence of tumor formation or a contributing factor to the development of this tumor, we analyzed the phenotype of Rb+/−p27−/− mice. We found that these mice developed pituitary adenocarcinoma with loss of the remaining wild-type allele of Rb and a high-grade thyroid C cell carcinoma that was more aggressive than the disease in either Rb+/− or p27−/− mice. Importantly, we detected both pituitary and thyroid tumors earlier in the Rb+/−p27−/− mice. We therefore propose that Rb and p27 cooperate to suppress tumor development by integrating different regulatory signals.

Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin

The interface between apoptosis (programmed cell death) and the cell cycle is essential to preserve homeostasis and genomic integrity. Here, we show that survivin, an inhibitor of apoptosis over-expressed in cancer, physically associates with the cyclin-dependent kinase p34cdc2 on the mitotic apparatus, and is phosphorylated on Thr34 by p34cdc2-cyclin B1, in vitro and in vivo. Loss of phosphorylation on Thr34 resulted in dissociation of a survivin-caspase-9 complex on the mitotic apparatus, and caspase-9-dependent apoptosis of cells traversing mitosis. These data identify survivin as a mitotic substrate of p34cdc2-cyclin B1 and suggest that survivin phosphorylation on Thr34 may be required to preserve cell viability at cell division. Manipulation of this pathway may facilitate the elimination of cancer cells at mitosis.

PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells

PTEN/MMAC1/TEP1 is a tumor suppressor that possesses intrinsic phosphatase activity. Deletions or mutations of its encoding gene are associated with a variety of human cancers. However, very little is known about the molecular mechanisms by which this important tumor suppressor regulates cell growth. Here, we show that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN was mediated by its ability to block cell cycle progression in the G1 phase. Such an arrest correlated with a significant increase of the cell cycle kinase inhibitor p27KIP1 and a concomitant decrease in the activities of the G1 cyclin-dependent kinases. PTEN expression also led to the inhibition of Akt/protein kinase B, a serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27KIP1 and the cell cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, our studies suggest that the PTEN tumor suppressor modulates G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase inhibitor p27KIP1.

p53 Accumulation, defective cell proliferation, and early embryonic lethality in mice lacking tsg101

Functional inactivation of the tumor susceptibility gene tsg101 in NIH 3T3 fibroblasts results in cellular transformation and the ability to form metastatic tumors in nude mice. The N-terminal region of tsg101 protein is structurally similar to the catalytic domain of ubiquitin-conjugating enzymes, suggesting a potential role of tsg101 in ubiquitin-mediated protein degradation. The C-terminal domain of TSG101 can function as a repressor of transcription. To investigate the physiological function of tsg101, we generated a null mutation of the mouse gene by gene targeting. Homozygous tsg101−/− embryos fail to develop past day 6.5 of embryogenesis (E6.5), are reduced in size, and do not form mesoderm. Mutant embryos show a decrease in cellular proliferation in vivo and in vitro but no increase in apoptosis. Although levels of p53 transcripts were not affected in tsg101−/− embryos, p53 protein accumulated dramatically, implying altered posttranscriptional control of p53. In addition, transcription of the p53 effector, cyclin-dependent kinase inhibitor p21WAF-1/CIP-1, was increased 5- to 10-fold, whereas activation of MDM2 transcription secondary to p53 elevation was not observed. Introduction of a p53 null mutation into tsg101−/− embryos rescued the gastrulation defect and prolonged survival until E8.5. These results demonstrate that tsg101 is essential for the proliferative burst before the onset of gastrulation and establish a functional connection between tsg101 and the p53 pathway in vivo.

Streptogramin- and tetracycline-responsive dual regulated expression of p27Kip1 sense and antisense enables positive and negative growth control of Chinese hamster ovary cells

We constructed a dual regulated expression vector cassette (pDuoRex) whereby two heterologous genes can be independently regulated via streptogramin- and tetracycline-responsive promoters. Two different constructs containing growth-promoting and growth-inhibiting genes were stably transfected in recombinant Chinese hamster ovary (CHO) cells that express the streptogramin- and tetracycline-dependent transactivators in a dicistronic configuration. An optimally balanced heterologous growth control scenario was achieved by reciprocal expression of the growth-inhibiting human cyclin-dependent kinase inhibitor p27Kip1 in sense (p27Kip1S) and antisense (p27Kip1AS) orientation. Exclusive expression of p27Kip1S resulted in complete G1-phase-specific growth arrest, while expression of only p27Kip1AS showed significantly increased proliferation compared to control cultures (both antibiotics present), presumably by decreasing host cell p27Kip1 expression. In a second system, a derivative of pDuoRex encoding streptogramin-responsive expression of the growth-promoting SV40 small T antigen (sT) and tetracycline-regulated expression of p27Kip1 was stably transfected into CHO cells. Expression of sT alone resulted in an increase in cell proliferation, but the expression of p27Kip1 failed to provide the expected G1-specific growth arrest despite having demonstrated expression of the protein. This illustrates the difficulty in balancing the complex pathways underlying cell proliferation control through the expression of two functionally distinct genes involved in those pathways, and how a single-gene sense/antisense approach using pDuoRex can overcome this barrier to complete metabolic engineering control.

Selective association of the methyl-CpG binding protein MBD2 with the silent p14/p16 locus in human neoplasia

DNA methylation of tumor suppressor genes is a common feature of human cancer. The cyclin-dependent kinase inhibitor gene p16/Ink4A is hypermethylated in a wide range of malignant tissues and the p14/ARF gene located 20 kb upstream on chromosome 9p21 is also methylated in carcinomas. p14/ARF (ARF, alternative reading frame) does not inhibit the activities of cyclins or cyclin-dependent kinase complexes; however, the importance of the two gene products in the etiology of cancer resides in their involvement in two major cell cycle regulatory pathways: p53 and the retinoblastoma protein, Rb, respectively. Distinct first exons driven from separate promoters are spliced onto the common exons 2 and 3 and the resulting proteins are translated in different reading frames. Both genes are expressed in normal cells but can be alternatively or coordinately silenced when their CpG islands are hypermethylated. Herein, we examined the presence of methyl-CpG binding proteins associated with aberrantly methylated promoters, the distribution of acetylated histones H3 and H4 by chromatin immunoprecipitation assays, and the effect of chemical treatment with 5-aza-2′-deoxycytidine (5aza-dC) and trichostatin A on gene induction in colon cell lines by quantitative reverse transcriptase–PCR. We observed that the methyl-CpG binding protein MBD2 is targeted to methylated regulatory regions and excludes the acetylated histones H3 and H4, resulting in a localized inactive chromatin configuration. When methylated, the genes can be induced by 5aza-dC but the combined action of 5aza-dC and trichostatin A results in robust gene expression. Thus, methyl-CpG binding proteins and histone deacetylases appear to cooperate in vivo, with a dominant effect of DNA methylation toward histone acetylation, and repress expression of tumor suppressor genes hypermethylated in cancers.

Skp2 is oncogenic and overexpressed in human cancers

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin–protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G1 progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. Tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-RasG12V to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.

Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a

The Id family of helix–loop–helix (HLH) transcriptional regulatory proteins does not possess a basic DNA-binding domain and functions as a negative regulator of basic HLH transcription factors. Id proteins coordinate cell growth and differentiation pathways within mammalian cells and have been shown to regulate G1-S cell-cycle transitions. Although much recent data has implicated Id1 in playing a critical role in modulating cellular senescence, no direct genetic evidence has been reported to substantiate such work. Here we show that Id1-null primary mouse embryo fibroblasts undergo premature senescence despite normal growth profiles at early passage. These cells possess increased expression of the tumor-suppressor protein p16/Ink4a but not p19/ARF, and have decreased cyclin-dependent kinase (cdk) 2 and cdk4 kinase activity. We also show that Id1 is able to directly inhibit p16/Ink4a but not p19/ARF promoter activity via its HLH domain, and that Id1inhibits transcriptional activation at E-boxes within the p16/Ink4a promoter. Our data provide, to our knowledge, the first genetic evidence for a role for Id1 as an inhibitor of cellular senescence and suggest that Id1 functions to delay cellular senescence through repression of p16/Ink4a. Because epigenetic and genetic abrogation of p16/Ink4a function has been implicated in the evolution of several human malignancies, we propose that transcriptional regulation of p16/Ink4a may also provide a mechanism for the dysregulation of normal cellular growth controls during the evolution of human malignancies.