Hypophosphorylation Of Rb Protein Research Articles

Abstract 1635: Inhibition of FANCA Suppresses Breast Cancer Development by Regulating Cell Cycle Progression

Abstract FANCA is one of the 22 known Fanconi anemia (FA) pathway genes that are involved in interstrand crosslink (ICL) repair. We have also shown that and FANCA plays a role in double strand break repair by promoting strand annealing and exchange. In this report, we describe that FANCA expression in breast cancer is significantly elevated within TCGA databases and in patient tumors-derived sections on both transcription and translation levels and this elevation is associated with poor outcomes of the breast cancer patients. Importantly, we have found that overexpressing FANCA in breast cancer cell line MDA-MB-231 promotes tumor growth both in vitro and in vivo. We have also demonstrated that FANCA inactivation by knocking out or knocking down inhibits growth of MDA-MB-231 in vitro and in vivo by causing cell cycle arrest at G1 phase resulting from hypophosphorylation of Rb protein. Our RNA-seq data has indicated that E2F targets are also downregulated in FANCA knockout (KO) cells compared to the wildtype (WT) MDA-MB-231 cells, which is consistent with the G1/S cell cycle arrest and Rb hypophosphorylation. Intriguingly, heterozygous FANCA KO in a breast cancer mouse model MMTV-PyMT model has shown reduction of breast tumor formation and volume. Our goal is to elucidate the role of FANCA in breast cancer development and to evaluate the potential of FANCA as a target for breast cancer treatment. Citation Format: Liang Luo, Wenjun Liu, Fenghua Yuan, Fang Li, Anna Palovcak, Daniel Calkins, Karoline Briegel Briegel, Yan Li, Christian Mason, Zhao-Jun Liu, Sylvia Daunert, Yanbin Zhang. Inhibition of FANCA Suppresses Breast Cancer Development by Regulating Cell Cycle Progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1635.

Higd-1a regulates the proliferation of pancreatic cancer cells through a pERK/p27KIP1/pRB pathway.

Higd-1a/HIMP1-a/HIG1, a mitochondrial inner membrane protein, promotes cell survival under low glucose and hypoxic conditions. We previously reported that it interacts with Opa1, a factor involved in mitochondrial fusion, to regulate mitochondrial homeostasis. In the present study, we found that depletion of Higd-1a inhibited the proliferation of pancreatic cancer cells in vitro and in mice xenografts. Higd-1a knockdown did not itself lead to cell death but it caused cell cycle arrest through induction of p27KIP1 and hypo-phosphorylation of RB protein. Knockdown of Higd-1a also induced cellular senescence as shown by increased granularity and SA-β-galactosidase activity. We further showed that the mitochondrial stress induced by Higd-1a led to reduced ERK phosphorylation. Inhibition of the ERK pathway with U0126 induced p27KIP1 expression in the pancreatic cancer cells, confirming that the cell cycle retardation was the result of inhibition of the ERK pathway. Array analysis of human pancreatic cancers revealed that expression of Higd-1a was significantly elevated in pancreatic cancer tissues compared to normal tissue. Collectively, our results demonstrate that Higd-1a plays an important role in the proliferation of pancreatic cancer cells by regulating the pERK/p27KIP1/pRB signaling pathway.

Induction of senescence pathways in Kindler syndrome primary keratinocytes

Individuals with Kindler syndrome (KS) have loss-of-function mutations in the FERMT1 gene that encodes the focal adhesion component kindlin-1. The major clinical manifestation of KS is epidermal atrophy (premature skin ageing). This phenotypic feature is thought to be related to the decreased proliferation rate of KS keratinocytes; nevertheless, molecular mediators of such abnormal behaviour have not been fully elucidated. To investigate how kindlin-1 deficiency affects the proliferative potential of primary human keratinocytes. We serially cultivated nine primary KS keratinocyte strains until senescence and determined their lifespan and colony-forming efficiency (CFE) at each serial passage. The expression of molecular markers of stemness and cellular senescence were investigated by immunoblotting using cell extracts of primary keratinocyte cultures from patients with KS and healthy donors. In another set of experiments, kindlin-1 downregulation in normal keratinocytes was obtained by small interfering RNA (siRNA) technology. We found that KS keratinocytes exhibited a precocious senescence and strongly reduced clonogenic potential. Moreover, KS cultures showed a strikingly increased percentage of aborted colonies (paraclones) already at early passages indicating an early depletion of stem cells. Immunoblotting analysis of KS keratinocyte extracts showed reduced levels of the stemness markers p63 and Bmi-1, upregulation of p16 and scant amounts of hypophosphorylated Rb protein, which indicated cell cycle-arrested status. Treatment of normal human primary keratinocytes with siRNA targeting kindlin-1 proved that its deficiency was directly responsible for p63, Bmi-1 and pRb downregulation and p16 induction. Our data directly implicate kindlin-1 in preventing premature senescence of keratinocytes.

47 Chromosomal complexity in primary myelodysplastic syndromes (MDS) in Nagasaki atomic bomb survivors

null linage negative bone marrow cells, from the inducible pLVXpTuner-Green lentiviral vector, restored the erythroid differentiation to the level seen in wild type. Similar results were observed in mouse hematopoietic progenitor cell line (EML) engineered to express p15Ink4b using the inducible pTuner system. Notably, induction of p15Ink4b protein for 24 hours was sufficient to increase commitment of the EML cell line to erythroid lineage, assayed by primitive BFU-E colony formation and increased expression of erythroid linage specific genes. Of interest, increased expression of p15Ink4b was associated with increased expression of EpoR and Gata1 mRNA and protein, hypophosphorylated Rb protein, and decreased expression of myeloid lineage specific transcription factor PU.1 protein. Furthermore, p15Ink4b null mice show delayed recovery from chemically induced anemia assayed by colony formation in methylcellulose based media and flow-cytometric analysis of all hematopoietic progenitors frequency. In summary, our results implicated a novel function of p15Ink4b in the bifurcation of erythroid and myeloid differentiation, thereby contributing to the development of MDS and AML.

Effects of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone on the growth inhibition in human pulmonary adenocarcinoma Calu-6 cells

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we evaluated the in vitro effects of FCCP on the growth of Calu-6 lung cancer cells. FCCP inhibited the growth of Calu-6 cells with an IC 50 of approximately 6.64 ± 1.84 μM at 72 h, as shown by MTT. DNA flow cytometric analysis indicated that FCCP induced G1 phase arrest below 20 μM of FCCP. Treatment with FCCP decreased the level of CDKs and cyclines in relation to G1 phase. In addition, FCCP not only increased the p27 level but also enhanced its binding with CDK4, which was associated with hypophosphorylation of Rb protein. While transfection of p27 siRNA inhibited G1 phase arrest in FCCP-treated cells, it did not enhance Rb phosphorylation. FCCP also efficiently induced apoptosis. The apoptotic process was accompanied with an increase in sub-G1 cells, annexin V staining cells, mitochondria membrane potential (MMP) loss and cleavage of PARP protein. All of the caspase inhibitors (caspase-3, -8, -9 and pan-caspase inhibitor) markedly rescued the Calu-6 cells from FCCP-induced cell death. However, knock down of p27 protein intensified FCCP-induced cell death. Moreover, FCCP induced the depletion of GSH content in Calu-6 cells, which was prevented by all of the caspase inhibitors. In summary, our results demonstrated that FCCP inhibits the growth of Calu-6 cells in vitro. The growth inhibitory effect of FCCP might be mediated by cell cycle arrest and apoptosis via decrease of CDKs and caspase activation, respectively. These findings now provide a better elucidation of the mechanisms involved in FCCP-induced growth inhibition in lung cancer.

Combined Treatment of γ-Tocotrienol with Statins Induce Mammary Tumor Cell Cycle Arrest in G1

Statins and gamma-tocotrienol (a rare isoform of vitamin E) both inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase activity and display anticancer activity. However, clinical application of statins has been limited by high dose toxicity. Previous studies showed that combined statin and gamma-tocotrienol treatment synergistically inhibits growth of highly malignant +SA mammary epithelial cells in culture. To investigate the mechanism mediating this growth inhibition, studies were conducted to determine the effect of combination low dose gamma-tocotrienol and statin treatment on +SA mammary tumor cell cycle progression. Treatment with 0.25 microM simvastatin, lovastatin, mevastatin, 10 microM pravastatin or 2.0 microM gamma-tocotrienol alone had no effect, while combined treatment of individual statins with gamma-tocotrienol significantly inhibited +SA cell proliferation during the 4-day culture period. Flow cytometric analysis demonstrated that combined treatment induced cell cycle arrest in G1. Additional studies showed that treatment with 0.25 microM simvastatin or 2 microM gamma-tocotrienol alone had no effect on the relative intracellular levels of cyclin D1, CDK2, CDK4 and CDK6, but combined treatment caused a large reduction in cyclin D1 and CDK2 levels. Combined treatments also caused a relatively large increase in p27, but had no effect on p21 and p15 levels, and resulted in a large reduction in retinoblastoma (Rb) protein phosphorylation at ser780 and ser807/811. Similar effects were observed following combined treatment of gamma-tocotrienol with low doses of lovastatin, mevastatin and pravastatin. These findings demonstrate that combination low dose statin and gamma-tocotrienol treatment induced mammary tumor cell cycle arrest at G1, resulting from an increase in p27 expression, and a corresponding decrease in cyclin D1, CDK2, and hypophosphorylation of Rb protein. These findings suggest that combined treatment of statins with gamma-tocotrienol may provide significant health benefits in the treatment of breast cancer in women, while avoiding myotoxicity associated with high dose statin monotherapy.

Anti-Leukemic Effect of a Synthetic Compound, (±)Trans-Dihydronarciclasione (HYU-01) Via Cell Cycle Arrest and Apoptosis

Over the past several decades, there has been considerable effort in the synthesis of narciclasine, lycoricidine, and pancratistatin. These naturally occurring isocarbostryls are known to have potent antitumoral and antiviral effects. Among them, trans-dihydronarciclasine isolated from the Chinese medicinal plant, Zephyranthes candida, exhibits even higher potency than pancratistatin against several human cancer cell lines and murine P388 lymphocytic leukemia cell line. However, much remains to be known about antitumoral mechanism of this natural product. In addition, the effect of transdihydronarciclasine in human acute myeloid leukemia (AML) has been not elucidated. The present study was undertaken to investigate the effect of novel synthetic (±)trans-dihydronarciclasine compound (code name; HYU-01) in human acute myeloid leukemia (AML). Treatment of HYU-01 for 72 hr inhibited the proliferation of human AML cell lines as well as primary leukemic blasts from AML patients in a dose-dependent manner with IC50 ranging from 1×10−7M to 5×10−8M. To address the mechanism of the antiproliferative effect of HYU-01, cell cycle analysis was performed in HL-60 cells. DNA flow cytometric analysis indicated that HYU-01 (2.5×10−7M) efficiently induced G1 arrest. Analysis of cell cycle-related proteins demonstrated that expression levels of CDK2, CDK4, CDK6, cyclin E and cyclin A were decreased in a time-dependent manner, and expression of cyclin D1 was up-regulated. In contrast, the level of cyclin B was not altered. In addition, HYU-01 (2.5×10−7M, 72 hr) increased the expression level of the CDKI p27kip1 and markedly enhanced the binding of p27 with CDK2, CDK4, and CDK6 compared to HYU-01-untreated cells. Furthermore, the activity of CDK2-associated kinase was decreased, which resulted in the hypophosphorylation of Rb protein. HYU- 01 also induced the apoptosis in HL-60 cells. The apoptotic process was associated with increased Bax and decreased Bid, Bcl-XL and poly(ADP-ribose) polymerase (PARP), primary leukemic blasts from AML patients in a dose-dependent manner with IC50 ranging from 1×10−7M to 5×10−8M. To address the mechanism of the antiproliferative effect of HYU-01, cell cycle analysis was performed in HL-60 cells. DNA flow cytometric analysis indicated that HYU-01 (2.5×10−7M) efficiently induced G1 arrest. Analysis of cell cycle-related proteins demonstrated that expression levels of CDK2, CDK4, CDK6, cyclin E and cyclin A were decreased in a time-dependent manner, and expression of cyclin D1 was up-regulated. In contrast, the level of cyclin B was not altered. In addition, HYU-01 (2.5×10− 7M, 72 hr) increased the expression level of the CDKI p27kip1 and markedly enhanced the binding of p27 with CDK2, CDK4, and CDK6 compared to HYU-01-untreated cells. Furthermore, the activity of CDK2-associated kinase was decreased, which resulted in the hypophosphorylation of Rb protein. HYU-01 also induced the apoptosis in HL-60 cells. The apoptotic process was associated with increased Bax and decreased Bid, Bcl-XL and poly(ADP-ribose) polymerase (PARP), and activation of caspase-8, -9, and -3, and release of cytochrome C from mitochondria into cytosol. In addition, the apoptosis by HYU-01 was accompanied with the down-regulation of ERK and P90RSK. These results suggest that HYU-01 inhibit the proliferation of AML cells via triggering the apoptosis as well as the induction of p27 and the reduction of CDK2 activity leading to G1 arrest.

Contribution of extracellular signal-regulated kinases to the IL-1-induced growth inhibition of human melanoma cells A375

The role of ERK1/2 in the IL-1-induced growth inhibition was investigated using human melanoma A375-6 cells. A selective inhibitor of ERK1/2 pathway, PD98059 and a selective inhibitor of p38MAPK, SB203580 each alone significantly reversed the IL-1-induced growth inhibition of A375-6 cells. Co-treatment with PD98059 and SB203580 completely reversed the IL-1-induced growth inhibition. ERK1/2 was constitutively activated in A375-6 cells, and IL-1 further augmented ERK activation. Antiproliferative effect of IL-1 was attenuated by the expression of dominant negative form of ERK2. IL-1 induced cell cycle arrest in G0/G1 phase, expression of p21 and p27 proteins, and down-regulation of cyclin D/cyclin-dependent kinase (CDK) 2 and CDK4 activities. These effects of IL-1 were reversed by PD98059. PD98059 also reversed the IL-1-induced hypophosphorylation of RB protein (pRB) and down-regulation of E2F activity. These findings demonstrate that ERK1/2 contribute to the IL-1-induced growth inhibition through induction of CDK inhibitors, down-regulation of CDK activity, pRB phosphorylation and E2F activity.

A transcription factor of lipid synthesis, sterol regulatory element‐binding protein (SREBP)‐1a causes G1 cell‐cycle arrest after accumulation of cyclin‐dependent kinase (cdk) inhibitors

Sterol regulatory element-binding protein (SREBP)-1a is a unique membrane-bound transcription factor highly expressed in actively growing cells and involved in the biosynthesis of cholesterol, fatty acids, and phospholipids. Because mammalian cells need to synthesize membrane lipids for cell replication, the functional relevance of SREBP-1a in cell proliferation has been considered a biological adaptation. However, the effect of this potent lipid-synthesis activator on cell growth has never been explored. Here, we show that induction of nuclear SREBP-1a, but not SREBP-2, completely inhibited cell growth in inducible Chinese hamster ovary (CHO) cell lines. Growth inhibition occurred through G(1) cell-cycle arrest, which is observed in various cell types with transient expression of nuclear SREBP-1a. SREBP-1a caused the accumulation of cyclin-dependent kinase (cdk) inhibitors such as p27, p21, and p16, leading to reduced cdk2 and cdk4 activities and hypophosphorylation of Rb protein. In contrast to transactivation of p21, SREBP-1a activated p27 by enhancing stabilization of the protein through inhibition of SKP2 and KPC1. In vivo, SREBP-1a-expressing livers of transgenic mice exhibited impaired regeneration after partial hepatectomy. SREBP-1-null mouse embryonic fibroblasts had a higher cell proliferation rate than wild-type cells. The unexpected cell growth-inhibitory role of SREBP-1a provides a new paradigm to link lipid synthesis and cell growth.

Trans-10,cis-12, Not cis-9,trans-11, Conjugated Linoleic Acid Inhibits G1-S Progression in HT-29 Human Colon Cancer Cells

Commercial preparations of conjugated linoleic acid (CLA) contain both positional and geometric isomers of octadecadienoic acid, with cis-9,trans-11 CLA (c9t11) and trans-10,cis-12 CLA (t10c12) as the principal isomers. We showed previously that CLA reduced the incidence of colon tumors in rats treated with 1,2-dimethylhydrazine. In addition, our previous in vitro studies showed that t10c12 inhibited the growth of HT-29 and Caco-2 human colon cancer cells, whereas c9t11 had no effect on cell growth. In the present study, to examine the effects of the CLA isomers on cell cycle and cell cycle regulatory proteins, we treated HT-29 cells with various concentrations (0-4 micromol/L) of the individual CLA isomers. A DNA flow cytometric analysis revealed that t10c12 induced a G1 arrest, whereas c9t11 had no effect on the cell cycle. Western blot analysis of total cell lysates revealed no alteration in the protein expression of cyclin A, cyclin D, cyclin E, cyclin-dependent kinase (CDK) 2, or CDK4 due to t10c12 treatment. However, t10c12 substantially increased the protein expression and mRNA accumulation of the CDK inhibitor p21(CIP1/WAF1). The t10c12 isomer increased the association of p21(CIP1/WAF1) with CDK2 and proliferating cell nuclear antigen, but decreased the levels of phosphorylated retinoblastoma protein (Rb), with an increase in the levels of hypophosphorylated Rb protein. An in vitro kinase assay using histone H1 as a substrate showed that the activities of CDK2 were significantly decreased by t10c12. These results indicate that t10c12 exerts its growth inhibitory effects in colon cancer cells through the induction of G1 cell cycle arrest. The induction of p21(CIP1/WAF1) may be one of the mechanisms by which t10c12 inhibits cell cycle progression in HT-29 cells.

Bone morphogenetic protein-2 induces hypophosphorylation of Rb protein and repression of E2F in androgen-treated LNCaP human prostate cancer cells

Bone morphogenetic protein-2 (BMP-2), a multifunctional member of the transforming growth factor (TGF)-beta, superfamily, has powerful osteoinductive effects and causes cell cycle arrest in a variety of transformed cell lines. We have observed BMP-2-induced inhibition of cell proliferation in an androgen-dependent human prostate cancer cell line (LNCaP). To investigate the mechanism of inhibition of androgen-dependent growth by BMP-2, we examined the effect of dihydrotestosterone (DHT) and/or BMP-2 on cell cycle-related proteins in LNCaP cells. BMP-2 decreased the phosphorylation of retinoblastoma (Rb) protein induced by treatment with DHT. DHT-induced expression of cyclin A and cyclin-dependent kinase 2 (CDK2) protein was also inhibited by co-treatment with BMP-2. Furthermore, BMP-2 induced expression of p21(WAF1/CIP1), a CDK inhibitor. These results indicate that changes in expression of these proteins lead to modulation of the phosphorylation state of Rb. DHT-induced E2F-1 protein and mRNA expressions was also inhibited by BMP-2, suggesting that BMP-2 inhibits DHT-induced growth of LNCaP cells through a decrease in E2F protein expression and suppression of E2F activity by hypophosphorylation of Rb.

High Loss of Heterozygosity of 9p24 Region and Identification of BRM as A Candidate Tumor Suppressor Gene in Head and Neck Cancer

Biochemical and genetic studies has identified several ATP-dependent multiprotein complexes that are involved in the remodeling of chromatin during gene activation. The SWI/SNF complex is one of these molecules. Molecular pathological changes of several members of SWI/SNF complex, especially human brahma (hBRM) and brahma-related gene 1 (BRG1) were reported to be involved in carcinogenesis of human cancer. hBRM can bind and cooperate with hypophosphorylated RB protein in repressing E2F1 transcriptional activation in transient transfection studies. We analyzed the LOH of the short arm of chromosome 9 in 64 head and neck squamous cell carcinomas by using 13 highly polymorphic microsatellite markers and found two deletion hot spots at 9p21 and 9p24. P16 tumor suppressor gene is likely to be a target for the deletion of 9p21 region. When the map of 9p24 region was redefined, a possible tumor suppressor gene hBRM was identified. Therefore we prepared an hBRM specific microsatellite marker and found 67% deletion of this gene at 9p24 region. In RT-PCR analysis about 60% of tumor samples demonstrated reduced mRNA expression as compared to matched normal samples.

Saucernetin-7 isolated from Saururus chinensis inhibits proliferation of human promyelocytic HL-60 leukemia cells via G0/G1 phase arrest and induction of differentiation.

In the present study, we investigated the in vitro effect of saucernetin-7, which is a dineolignan isolated from Saururus chinensis, on the proliferation, cell cycle-regulation and differentiation of HL-60 human promyelocytic leukemia cells. Saucernetin-7 potently inhibited the proliferation of HL-60 cells in both a dose- and time-dependent manner with an IC50, approximately 5 microM. DNA flow-cytometry indicated that saucernetin-7 markedly induced a G1 phase arrest of HL-60 cells. Among the G1 phase cell cycle-related proteins, the levels of cyclin-dependent kinase (CDK)6 and cyclin D1 were reduced by saucernetin-7, whereas the steady-state levels of CDK2, CDK4, cyclin D2, cyclin D3 and cyclin E were unaffected. The protein and mRNA levels of a CDK inhibitor p21CIP1/WAF1, but not p27KIP1, were markedly increased by saucernetin-7 and p21CIP1/WAF1 induction is likely to occur at the transcriptional level because actinomycin D blocked this induction. In addition, saucernetin-7 markedly enhanced the binding of p21CIP1/WAF1 with CDK2 and CDK6, resulting in the reduced activity of both kinases and the hypophosphorylation of Rb protein. We furthermore suggest that saucernetin-7 is a potent inducer of the differentiation of HL-60 cells, based on observations such as a reduction of the nitroblue tetrazolium level, an increase in the esterase activities and phagocytic activity, morphology changes, and the expression of CD14 and CD66b surface antigens. In conclusion, the onset of saucernetin-7-induced the G0/G1 arrest of HL-60 cells prior to the differentiation is linked to a sharp up-regulation of the p21CIP1/WAF1 level and a decrease in the CDK2 and CDK6 activities. This is the first report demonstrating that saucernetin-7 potently inhibits the proliferation of human promyelocytic HL-60 cells via the G1 phase cell cycle arrest and differentiation induction.

Monensin-mediated growth inhibition in NCI-H929 myeloma cells via cell cycle arrest and apoptosis

Previously, we showed that monensin, Na+ ionophore, potently inhibited the growth of acute myelogenous leukemia and lymphoma cells. Here, we investigated the antiproliferative effect of monensin on human myeloma cell lines. Monensin significantly inhibited the proliferation of myeloma cell lines examined with IC50 of about 1 micro M. Cell cycle analysis indicated that monensin induced a G1 and/or a G2-M phase arrest in these cell lines. To address the mechanism of the antiproliferative effect of monensin, we examined the effect of this drug on cell cycle-related proteins in NCI-H929 cells. Monensin decreased the levels of CDK2, CDK6, cdc2, cyclin A, cyclin B1, cyclin D1 and cyclin E proteins but did not alter CDK4 protein. While p21 was increased by monensin, p27 was not. In addition, monensin markedly enhanced the binding of p21 with CDK6 and cdc2. Furthermore, the activities of CDK2- and CDK6-associated kinases were reduced in association with hypophosphorylation of Rb protein. The activity of cdc2-associated kinase was decreased, which was accompanied by reduction of cdc25C phosphatase. Also, monensin induced apoptosis in myeloma cells, as evidenced by annexin V binding assay and flow cytometric detection of sub-G1 DNA content. This apoptotic process was associated with down-regulation of Bcl-2, loss of mitochondria transmembrane potential (Deltapsim) and an increase of caspase-3 activity. In addition, monensin caused the up-regulation of ERK and p38 kinase activities. Taken together, these results have demonstrated for the first time that monensin potently inhibited the proliferation of human myeloma cell lines, especially NCI-H929 cells, via cell cycle arrest in association with p21 and apoptosis.

Monensin inhibits the growth of renal cell carcinoma cells via cell cycle arrest or apoptosis

Previously, we showed that monensin, Na+ ionophore, potently inhibited the growth of acute myelogenous leukemia and lymphoma cells. Here, we demonstrate that monensin inhibited the proliferation of renal cell carcinoma cells with IC50 of about 2.5 micro M. Monensin induced a G1 or a G2-M phase arrest in these cells. When we examined the effects of this drug on ACHN cells, monensin decreased the levels of CDK2, CDK6, cdc2, cyclin A and cyclin B1 proteins. p21 and p27 proteins were increased by monensin. In addition, monensin markedly enhanced the binding of p21 with CDK2 and the binding of p27 with CDK6. Furthermore, the activities of CDK2- and CDK6-associated kinase were reduced in association with hypophosphorylation of Rb protein. Monensin also induced the apoptosis in several renal cell carcinoma cells. Apoptotic process of Caki-2 cells was associated with the changes of Bcl-2, Bcl-XL, caspase-9, caspase-3, caspase-7 proteins as well as mitochondria transmembrane potential (DeltaPsim) loss. Taken together, these results demonstrate for the first time that monensin inhibits the growth of renal cell carcinoma cells via cell cycle arrest or apoptosis.

Monensin-mediated growth inhibition of SNU-C1 colon cancer cells via cell cycle arrest and apoptosis

Previously, we showed that monensin, Na+ ionophore, potently inhibited the growth of acute myelogenous leukemia and lymphoma cells. Here, we demonstrate that monensin inhibited the proliferation of solid tumor cells with IC50 of about 2.5 micro M. Monensin induced a G1 or a G2-M phase arrest in these cells. When we examined the effects of this drug on SNU-C1 cells, monensin decreased the levels of CDK2, CDK4, CDK6, cyclin D1 and cyclin A proteins. While p27 was increased by monensin, p21 was not. In addition, monensin markedly enhanced the binding of p27 with CDK2, CDK4 and CDK6. Furthermore, the activities of CDK2-, CDK4- and CDK6-associated kinase were reduced in association with hypophosphorylation of Rb protein. Monensin also induced apoptosis in solid tumor cells. Apoptotic process of SNU-C1 cells was associated with the changes of Bax, caspase-3 and mitochondria transmembrane potential (deltapsim). Taken together, these results demonstrated for the first time that monensin inhibited the growth of solid tumor cells, especially SNU-C1 cells, via cell cycle arrest and apoptosis.

Arsenic trioxide inhibits the growth of A498 renal cell carcinoma cells via cell cycle arrest or apoptosis

Previously, we showed that arsenic trioxide potently inhibited the growth of myeloma cells and head and neck cancer cells. Here, we demonstrate that arsenic trioxide inhibited the proliferation of all the renal cell carcinoma cell lines (ACHN, A498, Caki-2, Cos-7, and Renca) except only one cell line (Caki-1) with IC 50 of about 2.5–10 μM. Arsenic trioxide induced a G 1 or a G 2-M phase arrest in these cells. When we examined the effects of this drug on A498 cells, arsenic trioxide (2.5 μM) decreased the levels of CDK2, CDK6, cyclin D1, cyclin E, and cyclin A proteins. Although p21 protein was not increased by arsenic trioxide, this drug markedly enhanced the binding of p21 with CDK2. In addition, the activities of CDK2- and CDK6-associated kinase were reduced in association with hypophosphorylation of Rb protein. Arsenic trioxide (10 μM) also induced apoptosis in A498 cells. Apoptotic process of A498 cells was associated with the changes of Bcl- XL, caspase-9, caspase-3, and caspase-7 proteins as well as mitochondria transmembrane potential (Δ ψ m) loss. Taken together, these results demonstrate that arsenic trioxide inhibits the growth of renal cell carcinoma cells via cell cycle arrest or apoptosis.

MyoD induces apoptosis in the absence of RB function through a p21(WAF1)-dependent re-localization of cyclin/cdk complexes to the nucleus.

During differentiation of skeletal myoblasts, MyoD promotes growth arrest through the induction of the cdk inhibitor p21 and the accumulation of hypophosphorylated RB protein. Myoblasts lacking RB function fail to accomplish full differentiation and undergo apoptosis. Here we show that exogenous MyoD induces apoptosis in several cell backgrounds sharing RB inactivation. This process is associated with increased levels of cell cycle-driving proteins and aberrant cell cycle progression. The inability of MyoD to induce apoptosis in a p21-null background, highlights a requirement of p21 in RB-regulated apoptosis during myogenesis. This pro-apoptotic function of p21 cannot be exerted by simple p21 over-expression, but requires the co-operation of MyoD. We also suggest that the essential aspect of p21 activity involved in such a process is related to its ability to induce the nuclear accumulation and aberrant activity of cyclin/cdk complexes. These results establish a novel link between MyoD, p21 and RB during myogenesis, providing new insights into the antagonism between muscle differentiation and loss of RB function.

Monensin-mediated growth inhibition in acute myelogenous leukemia cells via cell cycle arrest and apoptosis.

Monensin, an Na(+) ionophore, regulates many cellular functions including apoptosis. However, there has been no report about the antitumoral effect of monensin on acute myelogenous leukemia (AML). Here, we investigated the antiproliferative effect of monensin on AML cells in vitro and in vivo. Monensin efficiently inhibited the proliferation of all of 10 AML cell lines, with IC(50) of about 0.5 microM. DNA flow cytometric analysis indicated that monensin induced a G(1) and/or a G(2)-M phase arrest in these cell lines. To address the mechanism of the antiproliferative effect of monensin, we examined the effect of monensin on cell cycle-related proteins in HL-60 cells. The levels of CDK6, cyclin D1 and cyclin A were decreased. In addition, monensin not only increased the p27 level but also enhanced its binding with CDK2. Furthermore, the activities of CDK2- and CDK6-associated kinases reduced by monensin were associated with hypophosphorylation of Rb protein. Monensin also induced apoptosis in AML cells including HL-60 cells. The apoptotic process of HL-60 cells was associated with changes in Bax, caspase-3, caspase-8 and mitochondria transmembrane potential (Deltapsi(m)). In particular, monensin (i.p. at a dose of 8 mg/kg thrice weekly) significantly reduced the tumor size of BALB/c mice that were inoculated s.c. with its derived cell line, WEHI-3BD cells (69% growth inhibition relative to control group; p < 0.05). Tumors from monensin-treated mice exhibited increased apoptosis, and these tumor were immunohistochemically more stained with Bax, Fas and p53 antibodies than control tumors. In conclusion, this is the first report that monensin potently inhibits the proliferation of AML cells.

Rb localization and phosphorylation kinetics correlate with the cellular phenotype of cultured breast adenocarcinoma cells.

Retinoblastoma protein (Rb) expression has been correlated with state of differentiation, proliferation rate, and metastatic potential in breast adenocarcinomas and established cell lines. These observations, based on immunoreactivity of total Rb rather than hypophosphorylated protein, do not address the relationship between functional Rb and indicators of an aggressive transformed cellular phenotype. We hypothesized that the distribution of functional Rb and the kinetics of Rb phosphorylation would differ between cell lines representing immortalized mammary epithelium (MCF10A), differentiated nonmetastatic mammary adenocarcinoma (MCF-7), and poorly differentiated, highly metastatic mammary adenocarcinoma (MDA-MB-231) and that these differences would be informative of the cellular phenotype. Direct immunofluorescence microscopy was used to compare qualitatively the subcellular localization of total and hypophosphorylated Rb protein in synchronized and asynchronous cells. This technique was also used to quantitatively assess the amounts of hypophosphorylated Rb throughout the cell cycle in these representative cell lines. Total Rb stained more prominently than hypophosphorylated Rb in the nucleus of all asynchronous cells. Rb phosphorylation was more rapid in MCF-7 cells than in MCF10A cells, whereas Rb dephosphorylation appeared deregulated in MDA-MB-231 cells. We conclude that assessment of hypophosphorylated Rb may be more useful than assessment of total Rb for the evaluation of transformed breast adenocarcinoma phenotypes.