HIF involvement in the regulation of rat Sertoli cell proliferation by FSH.

The final number of Sertoli cells reached during the proliferative periods determines sperm production capacity in adulthood. It is well known that FSH increases the rate of proliferation of Sertoli cells; however, little is known about the transcription factors that are activated by the hormone in order to regulate Sertoli cell proliferation. On the other hand, Hypoxia Inducible Factors (HIFs) are master regulators of cell growth. HIFs are dimers of HIF-ß and HIF-α subunits. Considering that HIF-ß is constitutively expressed, HIF transcriptional activity is regulated through the abundance of HIF-α subunits. To date, three HIF-α isoforms have been described. The association of the different HIF-α subunits with HIF-ß subunit constitutes three active transcription factors -HIF-1, HIF-2 and HIF-3- which interact with consensus hypoxia-response elements in the promoter region of target genes. Hypoxia has been classically considered the main stimulus that increases HIF transcriptional activity, however, regulation by hormones under normoxic conditions was also demonstrated. The aim of this work has been to investigate whether HIFs participate in the regulation of rat Sertoli cell proliferation by FSH. Sertoli cells obtained from 8-day old rats were cultured in the absence or presence of FSH. It has been observed that FSH increases HIF transcriptional activity and HIF-2α mRNA levels without modifying either HIF-1α or HIF-3α expression. Incubations with FSH have been also performed in the absence or presence of a pharmacological agent that promotes HIF-α subunit degradation, LW6. It has been observed that LW6 inhibits the FSH effect on proliferation, CCND1 expression and c-Myc transcriptional activity. Altogether, these results suggest that HIFs might be involved in the regulation of Sertoli cell proliferation by FSH.

Primary refractory diffuse large B cell lymphoma in the rituximab era.

PURPOSE OF REVIEW: The aim of this review is to describe the outcome of primary refractory diffuse large B cell lymphoma in the rituximab era and the different therapeutic options as well as new biological markers that could allow the pathologist to distinguish these cases at diagnosis. RECENT FINDINGS: Diffuse large B cell lymphoma outcome has been impressively improved since the introduction of rituximab in association with anthracycline-based chemotherapy; however, primary refractory patients still represent an unmet medical need. SUMMARY: If patients without relapse after 2 years from diagnosis have an outcome comparable to healthy individuals, primary refractory patients still represent 20% of the cases with a very poor overall survival. These cases are usually described as progressive patients during first line or patients reaching a nonadequate partial response or those relapsing within a year after reaching a response.

Regulation of cellular Cyclin D1 gene by arsenic is mediated through miR-2909.

Arsenic through its ability to regulate genes that link cell cycle control with apoptosis has been widely recognized to play a crucial role in oncogenomics. However, the molecular event by which arsenic affects such genes is far from clear. Here we provide reasonably good evidence to support the view that arsenic exposure to human PBMCs (peripheral blood mononuclear cells) at low concentrations results in the over-expression of miR-2909 within these cells. This over-expressed miR-2909 was found to regulate CCND1 (Cyclin D1) gene expression, within these cells by inducing splice-switching of tumor suppresser CYLD (Cylindromatosis) gene as well as modulation of SP1 (Specificity Protein 1) activity through the repression of KLF4 (Kruppel-like factor4) expression at the translational level. Arsenic dependent regulation of AATF (Apoptosis Antagonizing Transcription factor) and BCL3 (B-cell Lymphoma 3) were also found to be modulated through its capacity to induce miR-2909 expression. Based upon these observations, a novel epigenomic pathway was proposed which may not only be useful in understanding the paradoxical role of arsenic in oncogenomics but also may even be useful in devising various strategies for the treatment/prevention of tumors induced by arsenic.

Inhibition of SAPK2/p38 enhances sensitivity to mTORC1 inhibition by blocking IRES-mediated translation initiation in glioblastoma.

A variety of mechanisms confer hypersensitivity of tumor cells to the macrolide rapamycin, the prototypic mTORC1 inhibitor. Several studies have shown that the status of the AKT kinase plays a critical role in determining hypersensitivity. Cancer cells in which AKT activity is elevated are exquisitely sensitive to mTORC1 inhibitors while cells in which the kinase is quiescent are relatively resistant. Our previous work has shown that a transcript-specific protein synthesis salvage pathway is operative in cells with quiescent AKT levels, maintaining the translation of crucial mRNAs involved in cell-cycle progression in the face of global eIF-4E-mediated translation inhibition. The activation of this salvage pathway is dependent on SAPK2/p38-mediated activation of IRES-dependent initiation of the cyclin D1 and c-MYC mRNAs, resulting in the maintenance of their protein expression levels. Here, we show that both genetic and pharmacologic inhibition of SAPK2/p38 in glioblastoma multiforme cells significantly reduces rapamycin-induced IRES-mediated translation initiation of cyclin D1 and c-MYC, resulting in increased G(1) arrest in vitro and inhibition of tumor growth in xenografts. Moreover, we observed that the AKT-dependent signaling alterations seen in vitro are also displayed in engrafted tumors cells and were able to show that combined inhibitor treatments markedly reduced the mRNA translational state of cyclin D1 and c-MYC transcripts in tumors isolated from mice. These data support the combined use of SAPK2/p38 and mTORC1 inhibitors to achieve a synergistic antitumor therapeutic response, particularly in rapamycin-resistant quiescent AKT-containing cells.

The impact of bisphosphonates on the osteoblast proliferation and Collagen gene expression in vitro.

BACKGROUND: Bisphosphonates are widely used in the clinical treatment of bone diseases with increased bone resorption. In terms of side effects, they are known to be associated with osteonecrosis of the jaw (BONJ).The objective of this study was to evaluate the effect of bisphosphonates on osteoblast proliferation by cell count and gene expression analysis of cyclin D1 in vitro. Furthermore, the gene expression of the extracellular matrix protein collagen type I was evaluated. Nitrogen-containing and non-nitrogen-containing bisphosphonates have been compared on gene expression levels. METHODS: Human osteoblast obtained from hip bone were stimulated with zoledronate, ibandronate and clodronate at concentrations of 5 x 10-5M over the experimental periods of 1, 2, 5, 10 and 14 days. At each point in time, the cells were dissolved, the mRNA extracted, and the gene expression level of cyclin D1 and collagen type I were quantified by Real-Time RT-PCR. The gene expression was compared to an unstimulated osteoblast cell culture for control. RESULTS: The proliferation appeared to have been influenced only to a small degree by bisphosphonates. Zolendronate led to a lower cyclin D1 gene expression after 10 days. The collagen gene expression was enhanced by nitrogen containing bisphosphonates, decreased however after day 10. The non-nitrogen-containing bisphosphonate clodronate, however, did not significantly influence cyclin D1 and collagen gene expression. CONCLUSIONS: The above data suggest a limited influence of bisphosphonates on osteoblast proliferation, except for zoledronate. The extracellular matrix production seems to be initially advanced and inhibited after 10 days. Interestingly, clodronate has little influence on osteoblast proliferation and extracellular matrix production in terms of cyclin D1 and collagen gene expression.

Krüppel-like factor 4 mediates anti-proliferative effects of progesterone with G0/G1 arrest in human endometrial epithelial cells.

Activation of the progesterone receptor (PR) inhibits cell proliferation in various reproductive tissues. However, the molecular mechanisms underlying the regulation of cell proliferation by PR remain poorly understood. It is well established that Krüppel-like factor 4 (KLF4), a family of zinc fingercontaining transcription factors, induces cell cycle arrest in epithelial cells. In this study, we investigated whether KLF4 served as a target of PR activation during cell proliferation using human endometrial epithelial cells. PR agonists, progesterone and dienogest, were found to produce a lasting increase in the expression of KLF4 mRNA, followed by a decrease in cyclin D1 mRNA, and inhibit cell proliferation with G0/G1 arrest. KLF4 knockdown using KLF4 small interferingRNA abrogated the inhibition of cell proliferation by PR agonists. In addition, forced expression of KLF4 inhibited cyclin D1 promoter transactivation. These results suggest that PR agonists induce KLF4 expression and then inhibit cyclin D1 expression, and consequently inhibit cell proliferation in human endometrial epithelial cells. In terms of human reproductive tissue, KLF4 may be a factor concerning cell cycle, directly responsive to PR activation.

Oligomycin induced the proteasomal degradation of cyclin D1 protein.

We searched for compounds that affect the cyclin D1/retinoblastoma protein pathway from the in-house natural product library using a recombinant adenovirus with the Cre/loxP-regulated cyclin D1 overexpression system, and we found that oligomycin inhibited cell growth more effectively in cyclin D1-overexpressing SW480 cells than in control SW480 cells. We also found that oligomycin reduced the expression levels of cyclin D1 protein and that this reduction is, at least in part, mediated by Thr-286 phosphorylation-dependent proteasomal degradation.

Rottlerin: a multifaced regulator of keratinocyte cell cycle.

In this study we showed that Rottlerin (also called Kamala or Mallotoxin), a natural product purified from Mallotus phillippinensis, is a potent suppressor of human keratinocytes (HaCaT cell line) proliferation. Following Rottlerin treatment, Thymidine incorporation into DNA and re-epithelialisation in a scratch wound model was decreased. At the molecular level, Rottlerin hampered the NFkB activation process, causing loss of cyclin D1 and promoting, in a PKCdelta-dependent pathway, ERK activation, which, in turn induced the cell cycle inhibitor p21 Cip1/Kip1. The NFkB-dependent drop in cyclin D1, along with the PKCdelta/ERK-dependent induction of p21 Cip1/Kip1, is responsible for growth arrest. These results open the way to further investigation on the Rottlerin therapeutic potential against keratinocyte hyper-proliferative disorders.

Runx2 transcriptional activation of Indian Hedgehog and a downstream bone metastatic pathway in breast cancer cells.

Runx2, required for bone formation, is ectopically expressed in breast cancer cells. To address the mechanism by which Runx2 contributes to the osteolytic disease induced by MDA-MB-231 cells, we investigated the effect of Runx2 on key components of the "vicious cycle" of transforming growth factor beta (TGFbeta)-mediated tumor growth and osteolysis. We find that Runx2 directly up-regulates Indian Hedgehog (IHH) and colocalizes with Gli2, a Hedgehog signaling molecule. These events further activate parathyroid hormone-related protein (PTHrP). Furthermore, Runx2 directly regulates the TGFbeta-induced PTHrP levels. A subnuclear targeting deficient mutant Runx2, which disrupts TGFbeta-induced Runx2-Smad interactions, failed to induce IHH and downstream events. In addition, Runx2 knockdown in MDA-MB-231 inhibited IHH and PTHrP expression in the presence of TGFbeta. In vivo blockade of the Runx2-IHH pathway in MDA-MB-231 cells by Runx2 short hairpin RNA inhibition prevented the osteolytic disease. Thus, our studies define a novel role of Runx2 in up-regulating the vicious cycle of metastatic bone disease, in addition to Runx2 regulation of genes related to progression of tumor metastasis.

Downregulation of SS18-SSX1 expression by small interfering RNA inhibits growth and induces apoptosis in human synovial sarcoma cell line HS-SY-II in vitro.

Substantial evidence indicates that the characteristic SS18-SSX fusion gene may play an important role in synovial sarcoma development and progression. For obtaining better insights into the genetic alterations and molecular mechanisms involved in synovial sarcomas and for developing novel therapeutic strategies for this disease, we first examined the efficiency of small interfering RNAs (siRNAs) targeting the SS18-SSX1 fusion gene in knocking down its expression in the human synovial sarcoma cell line HS-SY-II, and then evaluated the effects of downregulation of this gene on apoptosis, apoptosis-related gene expression, growth regulatory proteins, and the growth of tumor cells in vitro. We observed a marked decrease (by more than 87.6%) in SS18-SSX1 expression levels in cells transfected with a plasmid expressing hairpin siRNA for this gene, which was accompanied by (i) reduction in protein levels of cyclin D1 and cyclin A, (ii) reduction in antiapoptotic protein Bcl-2 and activation of caspase 3/apoptosis, and (iii) growth inhibition of HS-SY-II cells in vitro. Our results demonstrate that siRNA targeting of SS18-SSX1 may have therapeutic potential in the treatment of synovial sarcomas.

Oncogenic signaling pathways activated in DMBA-induced mouse mammary tumors.

Only about 5% of human breast cancers can be attributed to inheritance of breast cancer susceptibility genes, while the balance are considered to be sporadic in origin. Breast cancer incidence varies with diet and other environmental influences, including carcinogen exposure. However, the effects of environmental carcinogens on cell growth control pathways are poorly understood. Here we have examined oncogenic signaling pathways that are activated in mammary tumors in mice treated with the prototypical polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA). In female FVB mice given 6 doses of 1 mg of DMBA by weekly gavage beginning at 5 weeks of age, all of the mice developed tumors by 34 weeks of age (median 20 weeks after beginning DMBA); 75% of the mice had mammary tumors. DMBA-induced mammary tumors exhibited elevated expression of the aryl hydrocarbon receptor (AhR), c-myc, cyclin D1, and hyperphosphorylated retinoblastoma (Rb) protein. Because of this, the activation of upstream regulatory pathways was assessed, and elements of the Wnt signaling pathway, the NF-kappa B pathway, and the prolyl isomerase Pin-1 were found to be frequently up-regulated in the tumors when compared to normal mammary gland controls. These data suggest that environmental carcinogens can produce long-lasting alterations in growth and anti-apoptotic pathways, leading to mammary tumorigenesis.

JAK-STAT signaling involved in phorbol 12-myristate 13-acetate- and dimethyl sulfoxide-induced 2'-5' oligoadenylate synthetase expression in human HL-60 leukemia cells.

The JAK-STAT signal transduction cascade participates in various cellular processes, including immune response, cell replication, differentiation and oncogenesis. Here, we report that this cascade is induced in two human myeloid HL-60 leukemia cell variants by the granulocyte differentiation inducer dimethyl sulfoxide (DMSO) and macrophage differentiation inducer phorbol 12-myristate 13-acetate (PMA). DMSO and PMA also induced the expression and catalytic activity of 2'-5' oligoadenylate synthetase (2-5A synthetase), a known interferon (IFN) inducible enzyme. The HL-60 cell variants included HL-205, which is susceptible to DMSO- and PMA-induced differentiation, and HL-525, which is susceptible to DMSO- but not to PMA-induced differentiation. Treatment of HL-205 and HL-525 cells with DMSO and HL-205 cells with PMA-induced JAK1 phosphorylation, JAK1/STAT1 association, formation of STAT1-STAT2 heterodimers, and the binding of the active IFN stimulating growth factor 3 (ISGF3) to the IFN-stimulated response element (ISRE) fragment isolated from the 2-5A synthetase promoter. These events were either reduced or absent in the resistant HL-525 cells treated with PMA. Taken together, our data implicate the above signaling cascade in DMSO- and PMA-induced 2-5A synthetase expression and catalytic activity in the HL-60 cell system.

Microarray analysis of altered gene expression in murine fibroblasts transformed by nickel(II) to nickel(II)-resistant malignant phenotype.

B200 cells are Ni(II)-transformed mouse BALB/c-3T3 fibroblasts displaying a malignant phenotype and increased resistance to Ni(II) toxicity. In an attempt to find genes whose expression has been altered by the transformation, the Atlas Mouse Stress/Toxicology cDNA Expression Array (Clontech Laboratories, Inc., Palo Alto, CA) was used to analyze the levels of gene expression in both parental and Ni(II)-transformed cells. Comparison of the results revealed a significant up- or downregulation of the expression of 62 of the 588 genes present in the array (approximately 10.5%) in B200 cells. These genes were assigned to different functional groups, including transcription factors and oncogenes (9/14; fractions in parentheses denote the number of up-regulated versus the total number of genes assigned to this group), stress and DNA damage response genes (11/12), growth factors and hormone receptors (6/9), metabolism (7/7), cell adhesion (2/7), cell cycle (3/6), apoptosis (3/4), and cell proliferation (2/3). Among those genes, overexpression of beta-catenin and its downstream targets c-myc and cyclin D1, together with upregulated cyclin G, points at the malignant character of B200 cells. While the increased expression of glutathione (GSH) synthetase, glutathione-S-transferase A4 (GSTA4), and glutathione-S-transferase theta (GSTT), together with high level of several genes responding to oxidative stress, suggests the enforcement of antioxidant defenses in Ni-transformed cells.

Hyperphosphorylation of extracellular regulated kinase 2 (ERK2) and inhibition of JNK2 phosphorylation are associated with increased S-phase during transformation of Syrian hamster embryo cells by Malachite Green.

Malachite Green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by MG. In this study, we have studied the mitogen activated protein (MAP) kinase signal transduction pathway in preneoplastic cells induced by MG. Western blots of MG induced preneoplastic cells showed no phosphorylation of ERK1, an increased phosphoactive ERK2 associated with a decreased expression of phosphoactive JNK2. However, total forms of ERKs, JNKs and p38 Kinases showed similar levels of expression in control and preneoplastic SHE cells. Indirect immunofluorescence studies have shown a distinct nuclear localisation of phosphoactive ERKs in MG induced preneoplastic cells. Flow cytometric analysis showed an increase of S-phase cells in preneoplastic cells compared to control SHE cells. The present study indicates that hyperphosphorylation of ERK2, decreased JNK2 phosphorylation and an increase in S-phase cells seems to be the early changes associated with the MG induced malignant transformation of SHE cells in primary culture.

Estradiol induces proliferation of keratinocytes via a receptor mediated mechanism.

In this study, we investigated the effects of estradiol on the proliferation of neonatal keratinocytes, the expression of estrogen receptor isoforms, and the signaling mechanisms by which estradiol mediates cell growth. We demonstrate that estradiol binds neonatal keratinocytes with high affinity (Kd=5.2nM) and limited capacity (Bmax of 14.2fmol/mg of protein), confirming the presence of estrogen binding sites. Using specific antibodies, we demonstrate that keratinocytes express both estrogen receptor (ER)-alpha and ER-beta. At physiological concentrations, estradiol up-regulates the level of ER-alpha receptors in keratinocytes and induces keratinocyte proliferation. The proliferative effect of estradiol requires the availability of functional estrogen receptors, as it is abrogated by anti-estrogen administration. Estradiol effect on keratinocyte proliferation is most likely mediated in part by activation of a nongenomic, membrane-associated, signaling pathway involving activation of the extracellular signal regulated kinases 1 and 2 and in part by the genomic signaling pathway through activation of nuclear receptors.

Down-regulation of c-myc and Cyclin D1 genes by antisense oligodeoxy nucleotides inhibits the expression of E2F1 and in vitro growth of HepG2 and Morris 5123 liver cancer cells.

A number of genetic interactions are involved in the control of cell cycle, but their role and nature have not been completely clarified. The knowledge of the behavior of these interactions in hepatocellular carcinoma, could optimize preventive and therapeutic strategies based on cell cycle restraint. We studied downstream events following c-MYC and CYCLIN D1 gene inhibition, by lipoplex-delivered MYC and CYCLIN D1 antisense oligodeoxy nucleotides (aODNM, aODND1), in in vitro cultured human HepG2 and rat Morris 5123 hepatoma cells. 0.5-20 micro M aODN(M) and aODND1 inhibited in vitro growth of both cell types. Scramble oligomer (SCR) and sense ODNs had no or relatively poor effect. Ten micromolar aODNM and aODND1, but not SCR, also induced a significant increase in the apoptotic index of HepG2 and 5123 cells, and inhibited colony formation in soft agar by HepG2 cells. Treatment of the cells with aODNM plus aODND1 had no additive effect on growth and apoptosis. aODNM and aODND1 induced >50% decrease in c-MYC and CYCLIN D1 gene expression, respectively, at both mRNA and protein level. The inhibition of gene expression by aODNs was highly specific, and SCR was without effect. The reduction in c-MYC and CYCLIN D1 expression by aODNs, was associated with a >50% decrease in E2F1 mRNA and protein production, without changes in CYCLIN A and CYCLIN E expression. These results suggest the involvement of both c-MYC and CYCLIN D1 on E2F1 gene function, and indicate that aODNM and aODND1 may inhibit hepatoma cell growth through down-regulation of the E2F1 gene. The inhibition of E2F1 gene expression by E2F1 aODN, was associated with strong growth restraint of HepG2 cells. Thus, interactions of c-MYC and CYCLIN D1 with E2F1 gene are essential for cell cycle activity in hepatoma cells, and their inhibition may have a therapeutic effect.

Beta-catenin/Tcf-1-mediated transactivation of cyclin D1 promoter is negatively regulated by thyroid hormone.

Cyclin D1 is an oncogenic cyclin frequently over-expressed in cancer. To examine the effect of thyroid hormone (T3) and its receptor (TR) on the transcription of cyclin D1 gene, we co-transfected the chloramphenicol acetyl-transferase (CAT) reporter plasmid containing cyclin D1 promoter together with the expression plasmids for TRbeta1 and wild-type or mutant beta-catenin (SA) into 293T cells. In the presence of T3, beta-catenin-dependent transactivation of cyclin D1 promoter was suppressed by co-transfection of TRbeta1. The suppression by T3/TRbeta1 was in a dose-dependent manner. The CAT reporter gene in which Tcf/Lef-1 sites were fused to heterologous promoter was also suppressed by T3/TRbeta1. Furthermore, inhibition of endogenous wild-type beta-catenin by T3/TRbeta1 was observed in SW480 colon carcinoma cells with mutation of the adenomatous polyposis coli gene. These results indicate that the T3-bound TR inhibits the transcription of cyclin D1 through the Tcf/Lef-1 site, which is positively regulated by the Wnt-signaling pathway.

Cell cycle control by the thyroid hormone in neuroblastoma cells.

The thyroid hormone (T3) blocks proliferation and induces differentiation of neuroblastoma N2a-beta cells that overexpress the beta 1 isoform of the T3 receptor. An element in the region responsible for premature termination of transcription mediates a rapid repression of c-myc gene expression by T3. The hormone also causes a decrease of cyclin D1 gene transcription, and is able to antagonize the activation of the cyclin D1 promoter by Ras. In addition, a strong and sustained increase of the levels of the cyclin kinase inhibitor (CKI) p27(Kip1) are found in T3-treated cells. The increased levels of p27(Kip1) lead to a marked inhibition of the kinase activity of the cyclin-CDK2 complexes. As a consequence of these changes, retinoblastoma proteins are hypophosphorylated in T3-treated N2a-beta cells, and progression through the restriction point in the cell cycle is blocked.

Tectorigenin, an isoflavone of Pueraria thunbergiana Benth., induces differentiation and apoptosis in human promyelocytic leukemia HL-60 cells.

Cytotoxic effects of six isoflavonoids, tectorigenin, glycitein, tectoridin, glycitin, 6''-O-xylosyltectoridin, and 6''-O-xylosylglycitin isolated from the flower of Pueraria thunbergiana Benth. together with genistein, a known differentiation and apoptosis inducer, were examined. Among these isoflavonoids, tectorigenin and genistein exhibited cytotoxicity against various human cancer cells; glycitein showed only mild cytotoxicity. These results suggest that the isoflavone structure and 5-hydroxyl group are crucial for the cytotoxic properties and that glycosides are inactive. Moreover, tectorigenin induced differentiation of human promyelocytic leukemia HL-60 cells to granulocytes and monocytes/macrophages, and caused apoptotic changes of DNA in the cells, as did genistein. Tectorigenin also inhibited autophosphorylation of epidermal growth factor (EGF) receptor by EGF and decreased the expression of Bcl-2 protein, with less activity than genistein. From these results, tectorigenin may be a possible therapeutic agent for leukemia.