Increased Cell Cycle Progression Research Articles

Loss of Nuclear p27 Expression and Its Prognostic Role in Relation to Cyclin E and p53 Mutation in Gastroenteropancreatic Neuroendocrine Tumors

Gastroenteropancreatic neuroendocrine tumors (GEP-NET) are classified by the WHO, yet its prognostic value needs to be confirmed. Therefore, we aimed to determine the prognostic role of cell cycle key regulatory genes p53, p27kip1 (p27), and cyclin E in this tumor entity. Tumor specimen from 89 patients with a complete follow-up were studied immunohistochemically for p27 and cyclin E expression and for p53 mutations. The functional relevance of p27 was evaluated in the neuroendocrine cell lines BON1 (human) and INS1 (rat) by the use of small interfering RNA. Twenty-six of 29 benign, well-differentiated endocrine tumors (WHO class 1) showed a high expression (> 50%) of p27, whereas all 10 poorly differentiated endocrine carcinomas (WHO class 3) displayed a low expression of p27. Metastatic well-differentiated endocrine carcinomas (WHO class 2) showed a low p27 expression in 20 of 50 (40%) patients, which conferred a poor prognosis (median survival, 57 versus 140 months; P = 0.037). This prognostic dichotomy was improved by the use of a combination of p27 and cyclin E (high cyclin E/low p27 versus low cyclin E/high p27: median survival 53 months versus not reached; P = 0.0044). p53 mutations were rare (1 of 10 poorly differentiated endocrine carcinomas). Loss of p27 and overexpression of cyclin E play a critical role in the aggressiveness of gastroenteropancreatic neuroendocrine tumors. This coincides with increased cell cycle progression. We propose a discussion whether to incorporate the immunohistochemical expression of p27 into a revised classification to individualize therapeutic strategies in this tumor entity.

IL-21 induces in vivo immune activation of NK cells and CD8+ T cells in patients with metastatic melanoma and renal cell carcinoma

PurposeHuman interleukin-21 (IL-21) is a class I cytokine previously reported in clinical studies on immune responsive cancers. Here we report the effects of systemic IL-21 therapy on the immune system in two phase 1 trials with this novel cytokine.Experimental designRecombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100 μg/kg using two planned treatment regimens: thrice weekly for 6 weeks (3/week); or once daily for five consecutive days followed by nine dose-free days (5 + 9). The following biomarkers were studied in peripheral blood mononuclear cells (PBMC) during treatment: phosphorylation of STAT3, alterations in the composition of leukocyte subsets, ex vivo cytotoxicity, expression of effector molecules in enriched CD8+ T cells and CD56+ NK cells by quantitative RT-PCR, and gene array profiling of CD8+ T cells.ResultsEffects of IL-21 were observed at all dose levels. In the 5 + 9 regimen IL-21 induced a dose dependent decrease in circulating NK cells and T cells followed by a return to baseline in resting periods. In both CD8+ T cells and CD56+ NK cells we found up-regulation of perforin and granzyme B mRNA. In addition, full transcriptome analysis of CD8+ T cells displayed changes in several transcripts associated with increased cell cycle progression, cellular motility, and immune activation. Finally, cytotoxicity assays showed that IL-21 enhanced the ability of NK cells to kill sensitive targets ex vivo.ConclusionsIL-21 was biologically active at all dose levels administered with evidence of in vivo NK cell and CD8+ T cell activation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-008-0479-4) contains supplementary material, which is available to authorized users.

TRAIL induces proliferation of human glioma cells by c-FLIPL-mediated activation of ERK1/2.

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in TRAIL-sensitive human malignant glioma cells. We show for the first time that TRAIL stimulates cell growth in TRAIL-resistant glioma cells. TRAIL-induced cell growth in resistant cells occurred through increased cell cycle progression as determined by flow cytometry and Western blot analysis of retinoblastoma protein phosphorylation. Western blot analysis of TRAIL-treated resistant cells revealed phosphorylation of ERK1/2 proteins and in vitro kinase analysis confirmed the activation of the ERK1/2 kinases. Inhibition of MEK1 eliminated both TRAIL-induced ERK1/2 activation and cell proliferation. In addition, siRNA inhibition of c-FLIP expression eliminates TRAIL-induced ERK1/2 activation and proliferation. Furthermore, overexpression of c-FLIP(L) potentiates TRAIL-induced ERK1/2 activation and proliferation of resistant glioma cells. Our results have shown for the first time that TRAIL-induced ERK1/2 activation and proliferation of TRAIL-resistant human glioma cells is dependent upon the expression of the long form of the caspase-8 inhibitor c-FLIP(L).

RhoBTB2 (DBC2) Is a Mitotic E2F1 Target Gene with a Novel Role in Apoptosis

We have identified the RhoBTB2 putative tumor suppressor gene as a direct target of the E2F1 transcription factor. Overexpression of E2F1 led to up-regulation of RhoBTB2 at the level of mRNA and protein. This also occurred during the induction of E2F1 activity in the presence of cycloheximide, thus indicating that RhoBTB2 is a direct target. RNAi-mediated knockdown of E2F1 resulted in decreased RhoBTB2 protein expression, demonstrating that RhoBTB2 is a physiological target of E2F1. Because E2F1 primarily serves to transcribe genes involved in cell cycle progression and apoptosis, we explored whether RhoBTB2 played roles in either of these processes. We found RhoBTB2 expression highly up-regulated during mitosis, which was partially dependent on the presence of E2F1. Furthermore, overexpression of RhoBTB2 induced a short term increase in cell cycle progression and proliferation, while long term expression had a negative effect on these processes. We similarly found RhoBTB2 up-regulated during drug-induced apoptosis, with this being primarily dependent on E2F1. Finally, we observed that knockdown of RhoBTB2 levels via siRNA delayed the onset of drug-induced apoptosis. Collectively, we describe RhoBTB2 as a novel direct target of E2F1 with roles in cell cycle and apoptosis.

Apelin-induced vascular smooth muscle cell proliferation: the regulation of cyclin D1

Apelin is the endogenous ligand of the G protein-coupled receptor, APJ. Vascular smooth muscle cells express both apelin and APJ, which are important regulatory factors in the cardiovascular and nervous systems. Importantly, APJ is also involved in the pathogenesis if HIV-1 infection. We investigated whether vascular smooth muscle cell proliferation was regulated through an apelin-pERK1/2-cyclin D1 signal transduction pathway. Apelin-13 significantly stimulated vascular smooth muscle cell proliferation and increased cell cycle progression. Apelin-13 a decreased the proportion of cell in the G0/G1 phase while increasing the number of cells in S phase. Apelin-13 also increased the levels of cyclin D1, cyclin E and pERK1/2. Treatment of cells with the MEK inhibitor PD98059 attenuated the apelin-3-induced pERK1/2 activation. Similarly, treatment with PD98059 partially diminished the apelin-13-induced expression of cyclin D1 and vascular smooth muscle cell proliferation. Taken together, these data established that apelin-13 stimulates vascular smooth muscle cell proliferation by promoting the G1-S phase transition, and that this effect is mediated in part by an apelin-pERK1/2-cyclin D1 signal cascade.

Clinical, Morphological, and Biochemical Correlates of Head Circumference in Autism

Head growth rates are often accelerated in autism. This study is aimed at defining the clinical, morphological, and biochemical correlates of head circumference in autistic patients. Fronto-occipital head circumference was measured in 241 nonsyndromic autistic patients, 3 to 16 years old, diagnosed according to DSM-IV criteria. We assessed 1) clinical parameters using the Autism Diagnostic Observation Schedule, Autism Diagnostic Interview-Revised, Vineland Adaptive Behavioral Scales, intelligence quotient measures, and an ad hoc clinical history questionnaire; 2) height and weight; 3) serotonin (5-HT) blood levels and peptiduria. The distribution of cranial circumference is significantly skewed toward larger head sizes (p < .00001). Macrocephaly (i.e., head circumference >97th percentile) is generally part of a broader macrosomic endophenotype, characterized by highly significant correlations between head circumference, weight, and height (p < .001). A head circumference >75th percentile is associated with more impaired adaptive behaviors and with less impairment in IQ measures and motor and verbal language development. Surprisingly, larger head sizes are significantly associated with a positive history of allergic/immune disorders both in the patient and in his/her first-degree relatives. Our study demonstrates the existence of a macrosomic endophenotype in autism and points toward pathogenetic links with immune dysfunctions that we speculate either lead to or are associated with increased cell cycle progression and/or decreased apoptosis.

E1AF promotes breast cancer cell cycle progression via upregulation of Cyclin D3 transcription

E1AF transcription factor, a member of Ets family, is deregulated in many tumors and widely known to play critical roles in tumor metastasis via directly binding to the promoter of genes involved in tumor migration and invasion. Here, we found that E1AF overexpression promoted breast cancer cell cycle progression and growth in vivo as well as the transcription of cell cycle-related protein Cyclin D3. And, the interference of Cyclin D3 expression by transfecting with Cyclin D3 RNAi inhibited the positive role of E1AF in cell cycle progression. We further showed that decreasing the expression of E1AF by E1AF RNAi reduced Cyclin D3 transcription and expression, and inhibited cell cycle progression that was abrogated by Cyclin D3 overexpression. Taken together, E1AF increases cell cycle progression via upregulation of Cyclin D3 transcription, which elicits a new mechanism of breast cancer growth and a new mechanism of Cyclin D3 transcription.

Cdc42 Is Critical for Multi-Lineage Blood Cell Development from Hematopoietic Stem and Progenitor Cells.

Cdc42 is a member of the Rho GTPase family regulating multiple functions in eukaryotic cells. However, its involvement in blood cell development is not well studied. In our recent work using an inducible, conditional gene targeted mouse model (Mx-Cre;Cdc42loxP/loxP), we have shown that Cdc42 is important for hematopoietic stem cell (HSC) homing, lodging and engraftment, as well as maintenance of long-term HSC quiescence in the hematopoietic microenvironment. To define the contribution of Cdc42 to multi-lineage differentiation of HSCs, we have examined this mouse model for lymphoid, erythroid and myeloid lineage development. Firstly, although Cdc42 gene disruption in HSCs does not alter the overall bone marrow (BM) cellularity, it affects the frequency of multipotent progenitor cells (Table). The number of common lymphoid progenitors (CLPs) remain unchanged, but the number of phenotypic common myeloid progenitors (CMP) decreases, the number of phenotypic granulocytic myeloid progenitors (GMP) increases, and the myeloerythroid progenitors (MEP) decreases, in the Cdc42−/− mice. Secondly, despite the normal CLP content in the BM, the cellularity of Cdc42−/− thymus and T-lymphoid populations in peripheral blood is markedly reduced (Table). The T-cell developmental defect is associated with a thymus-homing defect of the Cdc42−/− BM cells. Thirdly, the Cdc42−/− mice show severe anemia and splenomegaly that are associated with dramatically decreased BFU-E and CFU-E activities of the BM cells (Table). FACS analysis showed an accumulation of early (CD71hiTER119−/low) erythroid cells and a reduction of the more mature TER119hi populations of erythroid progenitors in the Cdc42−/− bone marrow (Table), suggesting that Cdc42-deficiency causes a block in erythroid differentiation. Finally, Cdc42-deficiency in the HSCs led to a myeloproliferative disorder (MPD) phenotype, characterized by significantly increased monocytes, granulocytes and myeloid progenitors in the peripheral blood (Table), massive deposit of Gr-1+/Mac-1+ myeloid cells in the BM, and infiltration by the myeloid cells into distal organs such as spleen, liver and lung. These effects are a consequence of increased cell cycle progression and cell expansion accompanying the markedly increased mobilization of the short-term HSCs and myeloid progenitors from the BM to the peripheral organs found in the Cdc42−/− mice. Taken together, our results show that in addition to an essential role in maintaining HSC quiescence in the bone marrow niche, the Rho GTPase Cdc42 is critically involved in regulating multi-lineage differentiation of HSCs, including lymphopoiesis, erythropoiesis and myelopoiesis, through distinct mechanisms.WTCdc42−/−BM cellularity (X106)113.0±15.2116.8±26.4CLP (X106)0.0223±0.01390.0390±0.0277CMP (X106)0.228±0.0930.077±0.029*GMP (X106)1.47±0.332.70±1.07*MEP (X106)0.16±0.0580.051±0.031*Thymus weight (X10−3g/g body weight)2.37±0.780.49±0.36***CD4+CD8− (/ul PB)614.3±340.7282.2±179.1*CD4−CD8+ (/ul PB)446.1±332.7181.2±56.7*BFU-E (/105 BM cells)36±8.914.4±3.7CFU-E (/105 BM cells)87.2±12.426.4±5.7*CD71+Ter119− cells in BM (Stage I)1.3%±0.06%3.4%±0.38%***CD71−Ter119+ cells in BM (StageII-IV)39.7%±10.8%7.8%±1.3%**Gr1/Mac1+ cells in BM47.8%±27.2%83.3%±8.9%***p<0.05 **p<0.01 ***p<0.001

The p53–Bcl-2 connection

The tumor suppressor p53 and the proto-oncogene Bcl-2 were two of the earliest identified cancer genes. Mutant p53 proteins were first discovered in transformed murine cell lines,1,2 whereas Bcl-2 translocations were first identified in human follicular lymphoma.3,4,5 Despite this shared cancer relevance, they were initially thought to have little else in common. p53 was proposed to activate cell cycle check-points,6 whereas Bcl-2 was shown to inhibit cell death. Additionally, Bcl-2 overexpression was found exclusively in hematopoietic cancers, whereas p53 mutations were primarily found in solid tumors. However, the last 15 years have witnessed the emergence of strong genetic and biochemical ties between these two proteins, and it has become increasingly evident that signaling between p53 and Bcl-2 is of fundamental importance to cancer biology. This News and Commentary will focus on the pathways connecting p53 with Bcl-2 and the deregulation of this signaling network during tumor development. p53 is a sequence-specific transcription factor that is activated by diverse forms of cellular stress.7 Early studies of p53 focused on the ability of tumor-derived p53 mutants to promote cell growth and transformation.8–10 Subsequently, p53 was characterized as an essential mediator of cell cycle arrest in response to diverse cellular stresses.11,12 The first suggestion that p53 could promote apoptosis came more than 10 years after its discovery from experiments in which p53 was introduced into a p53-deficient leukemia cell line. These experiments showed that enforced p53 expression could induce cell death in cells deprived of prosurvival cytokines.13 Subsequently, work in p53 knockout mice demonstrated that p53 activity was essential for radiation-induced death in thymocytes and chemotherapy-induced apoptosis in fibroblasts expressing deregulated oncogenes.14–16 Importantly, the ability of p53 to promote cell death could be directly linked to its tumor suppressive function. Development of certain tumors in p53 null mice was associated with decreased cell death rather than increased cell cycle progression.17,18 Additionally, certain tumor-derived p53 mutants were shown to be impaired for apoptosis induction, but capable of promoting cell cycle arrest.19,20 Early studies also indicated that the proapoptotic activity of p53 correlated with its ability to function as a transcription factor, as tumor-derived p53 mutants defective in their ability to bind DNA in a sequence-specific manner were also found to be impaired for apoptosis induction.21,22 Still, some studies suggested p53 may promote apoptosis through transcription-independent functions.23,24 Only in the last decade have the relevant p53 targets in apoptosis come into focus. It now appears that the primary action of p53 in apoptosis is to directly and indirectly regulate the activity of the Bcl-2 family proteins.

TLE Corepressors as Novel Candidate AML Tumor Suppressor Genes That Regulate Wnt Signaling, Cell Cycle Progression, and Myeloid Differentiation.

Deletion of the long arm of chromosome 9, del(9q), is one of the most common mutations associated with t(8;21) AML. Up to 50% of del(9q) AML is seen in association with t(8;21). The Runx1-ETO (AML1-ETO) fusion gene produced by t(8;21) is insufficient for leukemogenesis and loss of a critical gene(s) on chr9q apparently cooperates in leukemogenesis. We recently identified two members of the Groucho family of co-repressors, Transducin-like enhancer of split (TLE) 1 and TLE4, as candidate tumor suppressor genes, based on our mapping of the commonly deleted region in del(9q) AML. These proteins are known to inhibit Wnt signaling which has been implicated in hematopoietic stem cell renewal and they interact with hematopoietic transcription factors such as Runx1 and Pu.1. We have demonstrated that the expression of these two genes is specifically repressed in both del(9q) and t(8;21) AML samples. Knockdown of TLE1 or TLE4 mRNA using specific small interfering RNAs (siRNA) activates Wnt signaling in 293T cells as measured by TOPFLASH activity, while forced expression of TLE1 or 4 inhibits TOPFLASH. We show that siRNA against TLE4 using a specific siRNA increased cell cycle progression and cell division, while over-expression of TLE1 or TLE4 slowed cell cycle progression and lead to a pronounced growth disadvantage in THP-1 and HL60 myeloid cell lines. In addition to these effects on cell proliferation, these genes also affected myeloid cell differentiation. Over-expression of either TLE1 or TLE4 in monocytic THP-1 and promyelocytic HL-60 cell lines initiated myeloid differentiation as monitored by CD11b expression. These two genes have different effects on more terminal myeloid differentiation as TLE4, but not TLE1, was able to induce more terminal differentiation of THP-1 cells into monocytes as measured by CD14 expression, while TLE1 was able to induce the granulocytic marker CD15 in HL-60 cells. Furthermore, lentiviral delivery of siRNAs for either TLE1 or TLE4 to HL-60 cells inhibited induction of granulocytic and monocytic differentiation with all-trans retinoic acid (ATRA) or 1,25-dihydroxycholechalciferol (Vit.D3). TLE1 siRNA inhibited ATRA and Vit.D3 induced CD11b expression by more than 75%. While TLE4 siRNA decreased the induction of monocytic CD14 after Vit.D3 by 40%, TLE1 siRNA almost completely abrogated induction of CD14 with Vit.D3 or granulocytic CD15 with ATRA in HL-60 cells. Ex vivo culture of siRNA infected CD34 sorted human cord blood (CB) cells in semi-liquid media with differentiation inducing cytokines for seven days exhibited a decrease in the percentage of early CD33+ myeloid cells by 15% and 70% relative to controls in TLE1 and TLE4 siRNA infected CB cells, respectively. These results indicate that inhibition of TLE activity can promote cell cycle progression and inhibit myeloid differentiation. This study is the first to demonstrate a potential role for the TLEs in leukemogenesis and also indicates the TLEs may have non-redundant functions in myeloid differentiation. Understanding how various mutations work cooperatively to produce a malignant phenotype is one of the great challenges in oncology. The TLEs may represent an important cooperating mutation with Runx1-ETO in AML that links Wnt signaling and core binding protein transcription factors. Studies are currently underway to demonstrate this cooperativity.

Telomerase Promotes the Self-Renewal of Human Hematopoietic Progenitor Cells but Does Not Maintain Telomere Length.

Telomerase is a reverse transcriptase that adds telomeric sequences onto chromosome ends. During hematopoiesis, telomerase activity is high in proliferating hematopoietic stem and progenitor cells, but decreases sharply as cells differentiate. Approximately 90% of all human cancers are telomerase positive, including samples from AML patients. To understand the role of telomerase in human hematopoietic stem and progenitor cell proliferation and differentiation, and in the development of leukemia, we established stable long-term cultures expressing high levels of telomerase by retrovirus-mediated transduction of the h-TERT (catalytic subunit of telomerase) gene into AML1-ETO-expressing CD34+ (AE) cells. We have previously shown that these AE cells maintain CD34 expression and proliferate for 7 months in culture but gradually lose telomere length and are not immortal, providing a good model for studying the molecular mechanisms involved in telomere shortening. Strikingly, telomerase expression enhanced the self-renewal ability of the human hematopoietic progenitor cells, as shown by continuous replating ability in methylcellulose assays. We also detected a pronounced delay in the differentiation of the progenitor cells upon telomerase expression, and an expansion of the progenitor pool. Telomerase expression promoted proliferation due to increased cell cycle progression as well as a survival advantage. At the same time, these cells demonstrated a progressive decline in telomere length, despite telomerase activity equivalent to that detected in leukemia cell lines. We conclude that expression of hTERT expands the pool of hematopoietic progenitors but does not maintain long telomeres in human CD34+ cells. hTERT plays a critical role not only in telomere homeostasis for genetic stability but also in cellular proliferation, differentiation, and self-renewal, functions that may be part of hTERT involvement in leukemogenesis. These data open an important debate regarding the specific contribution of telomerase expression to the leukemic phenotype and the potential timing of this essential hit in the progression of the disease.

Regulation of nuclear Prointerleukin-16 and p27 Kip1 in primary human T lymphocytes

Prointerleukin-16 (Pro-IL-16) is an abundant, PDZ domain-containing protein expressed in the nucleus and cytoplasm of resting human T lymphocytes. We have previously shown that ectopic expression of Pro-IL-16 in Pro-IL-16-negative human Jurkat cells represses transcription of the F-box protein, Skp2, resulting in accumulation of the cyclin-dependent kinase inhibitor, p27 Kip1, and G0/G1 cell cycle arrest. The current studies demonstrate the kinetics of Pro-IL-16 and p27 Kip1 expression in activated normal human T lymphocytes. We correlate nuclear Pro-IL-16 loss with decreased p27 Kip1 expression, increased cell cycle progression, and proliferation. Conversely, we show that constitutive expression of Pro-IL-16 by retroviral infection of activated human T lymphocytes induces G0/G1 cell cycle arrest, inhibits proliferation, and is associated with increased levels of p27 Kip1. These findings implicate nuclear Pro-IL-16 as a cell cycle regulatory protein for human T lymphocytes.

Tyrosine kinase-mediated activation of NAD(P)H oxidase enhances proliferative capacity of diabetic vascular smooth muscle cells

To investigate a potential molecular basis for a link between diabetes and atherosclerosis, experiments were performed to determine the role of NAD(P)H oxidase in the enhanced proliferative capacity of vascular smooth muscle cells (VSMC) from OLETF rat, an animal model of type 2 diabetes. An enhanced proliferative response to 10% fetal bovine serum with an increased cell cycle progression from G 1 to S phase as well as an augmented superoxide generation with an increased NAD(P)H oxidase activity were observed in diabetic versus control VSMC. Both the enhanced proliferation and superoxide generation in diabetic VSMC were significantly attenuated not only by diphenyleneiodonium (10 μM) and apocynin (100 μM), NAD(P)H oxidase inhibitors but also by protein tyrosine kinase inhibitors such as genistein (100 μM) and AG 112 (100 μM). Furthermore, the enhanced NAD(P)H oxidase activity in diabetic VSMC was significantly attenuated by genistein and AG112, but not by daidzein (100 μM), a genistein analogue devoid of protein tyrosine kinase inhibitory properties. Based on these results, it is suggested that the enhanced proliferative capacity of diabetic VSMC is closely related to the activation of NAD(P)H oxidase that is induced through activation of protein tyrosine kinase.

Constitutive activation of the shh–ptc1 pathway by a patched1 mutation identified in BCC

Mutations in the transmembrane receptor patched1 (ptc1) are responsible for the majority of basal cell carcinoma (BCC) cases. Many of these mutations, including ptc1-Q688X, result in premature truncation of the ptc1 protein. ptc1-Q688X has been identified in patients with both BCC and nevoid basal cell carcinoma syndrome, an inheritable disorder causing a predisposition to cancer susceptibility. Here we describe a mechanism by which ptc1-Q688X causes constitutive cellular signaling. Cells expressing ptc1-Q688X demonstrate an increase in cell cycle progression and induce cell transformation. The ptc1-Q688X mutant enhances Gli1 activity, a downstream reporter of sonic hedgehog (shh)-ptc1 signaling, independent of shh stimulation. In contrast to wild-type ptc1, ptc1-Q688X fails to associate with endogenous cyclin B1. Expression of nuclear-targeted cyclin B1 derivatives promotes Gli1-dependent transcription, which correlates temporally with cyclin B1-cdk1 kinase activity. Coexpression of wild-type ptc1 with a nuclear-targeted cyclin B1 derivative, mutated to mimic constitutive phosphorylation, dramatically decreases Gli1 activity. In addition, the coexpression of this constitutively nuclear cyclin B1 derivative with ptc1-Q688X substantially enhances foci formation. These studies therefore describe a molecular mechanism for the aberrant activity of ptc1-Q688X that includes the premature activation of the transcription factor Gli1.

T-cadherin upregulation correlates with cell-cycle progression and promotes proliferation of vascular cells.

In vascular tissue, T-cadherin (T-cad) levels correlate with the progression of atherosclerosis, restenosis and tumour neovascularization. This study investigates whether T-cad influences proliferation of vascular cells. Cultures of human umbilical vein endothelial cells (HUVEC) and rat and human aortic smooth muscle cells (rSMC, hSMC) were used. T-cad was overexpressed in HUVEC and hSMC using an adenoviral expression system. In cultures released from G(1)/G(0) synchrony parallel immunoblot analysis of T-cad and cell cycle phase specific markers (p27(Kip1), cyclin D1, E2F1, PCNA, cyclin B) showed increased T-cad protein levels subsequent to entry into early S-phase with sustained elevation through S-and M-phases. T-cad was increased in G(2)/M-phase (colchicine) synchronized cultures. In FACS-sorted cell populations, expression of T-cad in S-and G(2)/M-phase was higher than G(1)/G(0)-phase. Compared with empty-and LacZ-vector infected controls, HUVEC and hSMC overexpressing T-cad exhibited increased proliferation as assessed in enumeration and DNA synthesis assays. Additionally, following release from G(1)/G(0) synchrony, HUVEC and hSMC overexpressing T-cad enter S-phase more rapidly. Flow cytometry after BrdU/propidium labelling confirmed increased cell cycle progression in T-cad overexpressing cells. In vascular cells, T-cad is dynamically regulated during the cell cycle and its expression functions in the promotion of proliferation. T-cad may facilitate progression of proliferative vascular disorders such as atherosclerosis, restenosis and tumour angiogenesis.

Overexpression of the Colony-Stimulating Factor (CSF-1) and/or Its Receptor c-fms in Mammary Glands of Transgenic Mice Results in Hyperplasia and Tumor Formation

A number of recent studies have suggested that the colony-stimulating factor (CSF-1) and its receptor c-fms may be involved in the development of mammary glands during lactation and breast cancer. To study the role of CSF-1 or its receptor in initiation of mammary tumorigenesis, we have generated two independent lines of transgenic mice that overexpress either CSF-1 or c-fms under the control of the mouse mammary tumor virus promoter. Mammary glands of the virgin CSF-1 transgenic mice show increased ductal branching, hyperplasia, dysplasia, and other preneoplastic changes, which are indicative of increased cellular proliferation. Similar changes were also evident in the mammary glands of the c-fms transgenic mice. These changes became more prominent with age and resulted in mammary tumor formation. Moreover, secondary events like dimethylbenz(a)anthracene treatment accelerated mammary tumor formation in these mice. Although the expression of estrogen receptor alpha was not significantly changed in either of the transgenic mouse strains, progesterone receptor levels was higher in both transgenic lines as compared with the nontransgenic littermates. Expression of G1 cyclins was prominently increased in the mammary glands of both the CSF-1 and c-fms transgenic lines, suggesting increased cell cycle progression in these strains. In addition, the proliferation marker proliferating cell nuclear antigen (PCNA) and the mitogen-responsive transcription factor c-jun were also increased as compared with the nontransgenic controls. These findings, along with the histological data, support the hypothesis that CSF-1 and its receptor are involved in the etiology of breast cancer.

TNF-α induced DNA damage in primary murine hepatocytes

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, usually arising from a background of chronic inflammatory disease. Tumor necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine produced in response to tissue injury, endotoxin exposure or infection and TNF-alpha signalling in hepatocytes is associated with an increase in oxidative stress. DNA is vulnerable to reactive oxygen species (ROS)-induced damage, which is highly mutagenic. Cells respond to DNA damage through the stabilisation of the tumor suppressor p53, which maintains genomic fidelity through induction of a cell cycle arrest in order to allow repair or elimination of the damaged cell through apoptosis. This study was carried out to determine if TNF-alpha caused oxidative DNA damage in primary cultures of murine hepatocytes and whether any damage would result in the induction of the tumor suppressor p53 and cell-cycle arrest. Using a modified Comet assay, to measure DNA damage we have demonstrated that TNF-alpha causes the formation of 8-oxo-deoxyguanosine (8-oxodG), an established marker of oxidative DNA damage, and a lesion associated with chronic hepatitis in human livers. In addition, the increase in DNA damage did not result in p53 stabilisation and TNF-alpha caused an increase in cell-cycle progression. We believe that this study indicates a possible putative role for TNF-alpha in the early stages of malignant transformation of hepatocytes.

Multifaceted roles of TNF‐α in myoblast destruction: A multitude of signal transduction pathways

In catabolic conditions, such as cancer cachexia, a balance favouring a cytokine environment culminates in muscle destruction. Utilising an in vitro model to mimic muscle wasting, we elucidate here the multifaceted roles that one such cytokine, TNF-alpha, invokes in the degeneration process. Treatment of C2 skeletal myoblasts with TNF-alpha not only suppresses morphological and biochemical differentiation, but following an initial wave of proliferation, and of survival (24 h), induces apoptosis. Investigating the mechanisms underlying these diverse actions of TNF-alpha, we demonstrate that cell replication is dependent on rapid and sustained activation of MAP kinase. Map kinase is not, however, central to the death process, which is associated with a progressive rise in caspase-8 activity, and is accompanied by sustained activation of JNK1 and transient activation of JNK2. Caspase inhibition caused a dose responsive reduction in cell death, while inhibition of the JNKs caused a significant increase in apoptosis. We further report that PI3 kinase is not involved in conferring early protection against TNF-alpha-induced death. By contrast, inhibition of NF-kappaB in the presence of TNF-alpha culminates in increased cell cycle progression, decreased gadd45beta expression and significant and precociously increased cell death, when compared with TNF-alpha alone. Our results begin to characterise the mechanisms underlying the acute mitogenic and anti-apoptotic roles of TNF-alpha, which appear to be defined by a balance between MAP kinase, Jun kinase (JNK), NF-kappaB and gadd45beta. They establish that inhibition of any one of these molecules, as may occur following caspase activation, could eliminate vital stem cells required for skeletal muscle regeneration during chronic catabolic conditions.

Identification of Transcription Factor KLF8 as a Downstream Target of Focal Adhesion Kinase in Its Regulation of Cyclin D1 and Cell Cycle Progression

Focal adhesion kinase (FAK) is an important mediator of integrin signaling in the regulation of cell adhesion, migration, survival, and proliferation. Here we report the identification of the transcription factor KLF8 as a target of FAK in cell cycle regulation. KLF8 is induced by FAK and decreased by FAK dominant-negative mutant ΔC14. Overexpression of KLF8 increases cell cycle progression, whereas inhibition of endogenous KLF8 by siRNA reduces it. Cyclin D1 promoter is identified as a target of KLF8, which is activated both directly by KLF8 binding to the GT box A and by an indirect mechanism through its repression of a potential inhibitory regulator of cyclin D1. Transcription activation of cyclin D1 by FAK requires both Ets family and KLF8 factors in a temporally differential manner. Together, our data provide further insights into molecular mechanism for FAK to regulate cell cycle progression.

JNK1 Physically Interacts with WW Domain-containing Oxidoreductase (WOX1) and Inhibits WOX1-mediated Apoptosis

Transient activation of c-Jun N-terminal kinase (JNK) promotes cell survival, whereas persistent JNK activation induces apoptosis. Bovine testicular hyaluronidase PH-20 activates JNK1 and protects L929 fibroblasts from staurosporine-mediated cell death. PH-20 also induces the expression of a p53-interacting WW domain-containing oxidoreductase (WOX1, also known as WWOX or FOR) in these cells. WOX1 enhances the cytotoxic function of tumor necrosis factor and mediates apoptosis synergistically with p53. Thus, the activated JNK1 is likely to counteract WOX1 in mediating apoptosis. Here it is demonstrated that ectopic JNK1 inhibited WOX1-mediated apoptosis of L929 fibroblasts, monocytic U937 cells, and other cell types. Also, JNK1 blocked WOX1 prevention of cell cycle progression. By stimulating cells with anisomycin or UV light, JNK1 became activated, and WOX1 was phosphorylated at Tyr(33). The activated JNK1 physically interacted with the phosphorylated WOX1, as determined by co-immunoprecipitation. Alteration of Tyr(33) to Arg(33) in WOX1 abrogated its binding interaction with JNK1 and its activity in mediating cell death, indicating that Tyr(33) phosphorylation is needed to activate WOX1. A dominant negative WOX1 was developed and shown to block p53-mediated apoptosis and anisomycin-mediated WOX1 phosphorylation but could not inhibit JNK1 activation. This mutant protein bound p53 but could not interact with JNK1, as determined in yeast two-hybrid analysis. Taken together, phosphorylation of JNK1 and WOX1 is necessary for their physical interaction and functional antagonism.