Presence Of Rb1 Research Articles

Clinical characteristics and treatment management of combined large cell neuroendocrine carcinoma, a subtype of large cell neuroendocrine carcinoma.

Combined large cell neuroendocrine carcinoma (CLCNEC) is a rare neuroendocrine carcinoma, accounting for approximately 10% of large cell neuroendocrine carcinoma (LCNEC). Mainly composed of coexisting adenocarcinoma components, with strong invasiveness and poor prognosis. The treatment regimen for CLCNEC mainly refers to complete surgical resection as the first choice in the early stage, while patients with stage II or higher require adjuvant treatment. At present, research on CLCNEC is mostly small sample and retrospective, and there is no consensus on whether molecular typing and treatment should be carried out. There is considerable controversy over whether it should be managed as small-cell lung cancer (SCLC) or non-small-cell lung cancer (NSCLC). Therefore, in order to solve the problem of confusion in the selection of treatment regimens for CLCNEC, while also considering the therapeutic effects, this article summarizes and analyzes previous studies, fully seeks evidence, and boldly proposes new therapeutic insights: the etoposide-platinum (EP) regimen serves as the basis for adjuvant therapy; In addition, SCLC/NSCLC-CLCNEC can be distinguished based on presence of RB1 and TP53 co-mutation, and targeted therapy or NSCLC type chemotherapy including platinum + gemcitabine or taxanes (NSCLC-GEM/TAX) can be used in combination or sequentially for NSCLC-CLCNEC.

Real-World comprehensive genomic profiling data for diagnostic clarity in pulmonary Large-Cell neuroendocrine carcinoma

BackgroundPulmonary large-cell neuroendocrine carcinoma (LCNEC) is an uncommon subtype of lung cancer believed to represent a spectrum of tumors sharing characteristics of both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Other groups have proposed genomic LCNEC subtypes, including small cell-like, non-small cell-like, and carcinoid-like subtypes. The primary goal of this study was to better define the NSCLC-like subtype with comprehensive genomic profiling (CGP). MethodsAn institutional database was queried to identify tissue specimens (TBx, N = 1,426) and liquid biopsies (LBx, N = 39) submitted for CGP during routine clinical care (8/2014 – 7/2023) with a disease ontology of LCNEC. TBx were profiled with FoundationOne® (F1) or F1CDx, using hybrid-capture technology to detect genomic alterations (GAs). Results1,426 LCNEC samples were genomically profiled. The presence of RB1 and TP53 genomic alterations (GAs) were used to define a SCLC-like subtype (n = 557). A carcinoid-like group was defined by the presence of MEN1 mutation in the absence of TP53 GAs (n = 25). The remaining 844 samples were compared to the SCLC-like group and GAs enriched relative to the SCLC-like samples with a false discovery rate (FDR) < 0.0001 were used to define a NSCLC-like group. These NSCLC-like subtype-defining GAs included SMARCA4, KRAS, FGF3/4/19, STK11, CDKN2A/B, MTAP, and CCND1. Under this schema, 530 samples were classified as NSCLC-like and 314 remained unclassified. ConclusionsLarge-scale CGP can better characterize biologically distinct molecular subtypes in LCNEC. Further studies to define how these molecular subtypes may help inform treatment decisions in this complex and challenging malignancy are warranted.

Abstract 1952: CDK4/6 inhibition radiosensitizes RB1 wild type triple negative breast cancers through impaired homologous recombination

Abstract Purpose: Cyclin-dependent kinase 4&amp;6 (CDK4/6) inhibitors have been approved for the treatment of metastatic, estrogen receptor positive (ER+) breast cancers, but there is growing interesting in CDK4/6 inhibition as a therapeutic strategy in other breast cancer subtypes, including triple negative breast cancer (TNBC). Previous studies have shown that CDK4/6 inhibition radiosensitizes ER+ breast cancers, but the interaction between CDK4/6 inhibition and radiation (RT) in TNBC is incompletely understood. Methods: Cellular viability was quantified 72 hours after drug treatment (in the absence of RT) to calculate a half maximal inhibitory concentration (IC50) value of proliferation. Radiation enhancement ratios (rER) and surviving fractions of cells after RT were calculated using clonogenic survival assays in RB1 wild type and mutant TNBC cell lines. Homologous recombination (HR) was assessed using RAD51 foci formation and a stable HR reporter system. G1 cell cycle arrest was quantified using propidium iodide-based flow cytometry. CRISPR-induced knockout of RB1 and transient siRNA-mediated knockdown of RB1 in TNBC cell lines was used in both clonogenic survival assays and immunofluorescence experiments. In vivo efficacy of CDK4/6 inhibition + RT was assessed using TNBC patient-derived xenograft models (PDX4664). Results: Although most TNBC cell lines are resistant to CDK4/6 inhibitor monotherapy (IC50 &amp;gt; 250nM) compared to ER+ cells, treatment with 250nM-1μM palbociclib radiosensitized RB1 wild type TNBC (MDA-MB-231, CAL-51, SUM-159, CAL-120; rER 1.08 – 2.22) but failed to radiosensitize RB1 mutant TNBC (CAL-851, MDA-MB-468; rER: 0.84 – 1.00). Radiosensitization of TNBC cell lines also occurred with short term ribociclib or abemaciclib pretreatment. At 6 and 16 hours following RT, significant suppression of RT-induced homologous recombination (HR) activity (RAD51 foci) was observed in RB1 wild type (p &amp;lt; 0.001) but not RB1 mutant (p &amp;gt; 0.05) TNBC cell lines. Cell cycle arrest after short term CDK4/6 inhibition was dependent on the presence of RB1. In addition, genetic knockdown of RB1 in RB1 wild type TNBC lead to a loss of CDK4/6 inhibitor-mediated HR suppression (p &amp;gt; 0.05) and diminished radiosensitization. Conclusions: In TNBC, CDK4/6 inhibition and RT leads to suppression of HR activity in an RB1-dependent manner. While ongoing studies seek to elucidate the role of RB1 in HR suppression in the context of CDK4/6 inhibitor-mediated radiosensitization of TNBC, our data suggests that CDK4/6 inhibition + RT could be a valuable clinical strategy to radiosensitize a wide range of breast cancer subtypes, including RB1 wild type TNBC. Citation Format: Andrea M. Pesch, Nicole Hirsh, Anna R. Michmerhuizen, Benjamin C. Chandler, Kari Wilder-Romans, Meilan Liu, Lori J. Pierce, James M. Rae, Corey W. Speers. CDK4/6 inhibition radiosensitizes RB1 wild type triple negative breast cancers through impaired homologous recombination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1952.

RB1 and TP53 co-mutations correlate strongly with genomic biomarkers of response to immunity checkpoint inhibitors in urothelial bladder cancer

BackgroundMuscle invasive urothelial bladder carcinoma (MIBC) present RB1 and TP53 somatic alterations in a variable percentage of tumors throughout all molecular subtypes. MIBCs with neuroendocrine features have a high response rate to immunity checkpoint inhibitors (ICIs). Whether the presence of somatic co-alterations in these 2 genes in MIBCs is relevant to their responsiveness to ICIs is not known.MethodsThe potential correlation of different genomic biomarkers of response to ICIs like tumor mutational burden (TMB), single nucleotide variants (SNV) predicted neoantigens, DNA damage response (DDR) genes, DNA somatic signatures and TILs infiltrate was explored in patients with somatic co-alterations in RB1 and TP53 (RB1&TP53) as compared with patients with no alterations in any (double wild type, DWT) or with alterations in just one of the 2 genes. The Cancer Genome Atlas (TCGA) pancancer BLCA dataset of cystectomy specimens (n = 407) with mutation, copy number alterations and transcriptomic (RNA sequencing) data as well as the IMVigor 210 study (n = 348) of metastatic urothelial bladder cancers treated with atezolizumab (PD-L1 inhibitor) with clinical response data containing transcriptomic (RNA sequencing), along with a subset (n = 274) with mutation and copy number data were used for this purpose. A novel tumor microenvironment metascore (TMM) was developed based in a LASSO regularized Cox model with predictive and prognostic ability.ResultsSamples with co-altered RB1&TP53: a) were enriched in immunity effectors (CD8 cytotoxic lymphocytes, NK cells) and display higher scores of a T cell inflamed signature; b) have a higher TMB, higher number of SNV predicted neoantigens and higher TILs fractions; c) have a higher number of DDR mutated and deep deleted DDR genes; d) have DNA somatic signatures 2 and 13 related to APOBEC mutagenesis. Using the IMVigor 210 dataset, RB1&TP53 samples had the highest response rate to atezolizumab and a strong correlation with TMB and TMM. The consensus molecular subtype classification in the IMVigor 210 dataset showed a significant correlation with both the response to treatment (p = 0.001, Chisquare) and the presence of RB1 and TP53 genomic alterations (p < 0.001, Chisquare).ConclusionsRB1&TP53 co-alterations are strongly associated with genomic biomarkers of response to ICIs in MIBCs.

Abstract B34: PD-0332991/palbociclib facilitates the physical interaction between RB1 and BIN1 to increase the vulnerability of pediatric osteosarcoma cells to PARP inhibition

Abstract Osteosarcoma (OS) is a sporadic bone malignancy that is diagnosed mainly in childhood and adolescence. Conventional genotoxic chemotherapy such as cisplatin and doxorubicin has been used as first-line therapy for pediatric OS patients. However, OS cells eventually acquire chemoresistance with poor prognosis. Once OS cells acquire chemoresistance, little or no therapeutic option is available. It is therefore critical to better understand genetic, epigenetic, and signaling alterations in OS cells, by which a novel mechanism-based therapeutic strategy to halt the development of OS may be established. Although there is no single genetic event leading to the development of OS, increased mortality, metastasis, and chemoresistance are frequently observed in pediatric OS patients deficient in the retinoblastoma 1 tumor suppressor protein (RB1), compared to RB1-positive OS cases. These facts suggest that the activation and maintenance of RB1 function may improve the efficacy of OS chemotherapy. In this study, we found that, particularly when RB1 is hypophosphorylated, RB1 physically interacts with the proapoptotic tumor suppressor BIN1 in the human OS cell line U2OS for growth suppression. The adenovirus type 5 E1A oncoprotein disrupted the physical RB1/BIN1 complex, which implies that the protein complex is functionally meaningful as potential anticancer machinery. Acting through the coiled-coil effector domain of BIN1, BIN1 physically interacted with the C pocket domain of RB1 (RB1-C) in vitro. Consistent with this, heterologous expression of BIN1 hindered the effect of serum stimulation on phosphorylation of RB1-C at serine 807/811, which is mediated by cyclin-dependent kinases 4 and 6 (CDK4/6). The transcriptional repression caused by a recombinant BIN1 protein fused with the DNA-binding domain of the yeast transcription factor GAL4 was counterbalanced by suberoylanilide hydroxamic acid (SAHA), a chemical inhibitor of histone deacetylases (HDAC), only in the presence of RB1. This suggests that the BIN1-dependent gene repression greatly requires an HDAC activity employed by RB1. As predicted, in vitro BIN1-induced cancer suppression was compromised by the cotransfection of RB1 short-hairpin RNA. Of note, PD-0332991/palbociclib, a U.S. FDA-approved chemical inhibitor of CDK4/6, which is known to induce hypophosphorylated RB1, stabilized the RB1/BIN1 complex in U2OS cells and markedly increased the susceptibility to olaparib, an FDA-approved inhibitor of poly(ADP-ribose) polymerase (PARP), in a manner dependent on BIN1 and RB1. Because U2OS cells do not carry the molecular features with BRCA1/2-deficient tumors or “BRCAness,” our study suggests a novel BRCAness-independent synthetic lethal possibility activated by the combination of PD-0332991/palbociclib and olaparib in pediatric OS cells. Citation Format: Watson P. Folk, Kelly Homlar, Daitoku Sakamuro. PD-0332991/palbociclib facilitates the physical interaction between RB1 and BIN1 to increase the vulnerability of pediatric osteosarcoma cells to PARP inhibition [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B34.

Functional characterization and crystal structure of thermostable amylase from Thermotoga petrophila, reveals high thermostability and an unusual form of dimerization

Thermostable α-amylases have many industrial applications and are therefore continuously explored from novel sources. We present the characterization of a novel putative α-amylase gene product (Tp-AmyS) cloned from Thermotoga petrophila. The purified recombinant enzyme is highly thermostable and able to hydrolyze starch into dextrin between 90 and 100°C, with optimum activity at 98°C and pH8.5. The activity increased in the presence of Rb1+, K1+ and Ca2+ ions, whereas other ions inhibited activity. The crystal structure of Tp-AmyS at 1.7Å resolution showed common features of the GH-13 family, however was apparently found to be a dimer. Several residues from one monomer interacted with a docked acarbose, an inhibitor of Tp-AmyS, in the other monomer, suggesting catalytic cooperativity within the dimer. The most striking feature of the dimer was that it resembled the dimerization of salivary amylase from a previous crystal structure, and thus could be a functional feature of some amylases.

Alternatively Spliced Tissue Factor As a New Therapeutic Target in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is marked by high expression of Tissue Factor (TF). Alternatively spliced TF (asTF) is a secreted TF form that signals non-proteolytically via β1/β3 integrins. asTF is abundant in PDAC lesions and its constitutive overexpression promotes tumor growth, metastatic spread, monocyte recruitment, and elevated procoagulant potential of PDAC cells and cell-derived microparticles. In this study, we determined if 1) delayed-onset overexpression of asTF yields a phenotype distinct from that obtained with constitutive overexpression; 2) asTF contributes to PDAC cell migration; and 3) monoclonal antibody-based targeting of tumor-derived asTF slows PDAC progression and/or asTF release into circulation in an orthotopic setting.Nude mice were orthotopically transplanted with human PDAC cells harboring a doxycycline (Dox)-inducible asTF transgene system (line Pt45P1/asTFi); mice received Dox (2µg/mL) in sucrose at day 1 (“Dox”), day 25 (“late Dox”), or sucrose alone (“no Dox”), and tumor progression was monitored in vivo over 7 weeks via SapC-DOPS imaging. Migration of Pt45P1/asTFi cells toward serum was assessed using laminin-coated transwell inserts. Plasma of PDAC tumor-bearing mice was assayed for asTF using a custom ELISA. Pt45P1 cells were resuspended in PBS containing anti-asTF rabbit monoclonal antibody Rb1 and implanted in a) nude mice; b) SCID mice, and c) SCID mice with TF levels reduced by 99% (SCID/TF-low) and tumor progression was monitored as above over 5 weeks. Paraffin-embedded tumor specimens were assessed for vessel density (isolectin B4 and/or anti-CD31 staining) and monocyte/macrophage infiltration (anti-F4/80 and/or anti-CD206 staining).“Late Dox” mice developed tumors comparable in size and vessel density to those in “Dox” mice, yet less infiltrated with macrophages (Dox: 70±22 cells/LPF, late Dox: 51±17 cells/LPF, p=0.009); tumor spread was significantly reduced in “late Dox” mice compared to “Dox” mice (7.52e+7 vs 1.22e+8 P/s/mm2, p=0.01). “Dox” mice also had high levels of asTF in circulation (~1 ng/mL), which did not differ significantly from those in “late Dox” mice. The PDAC cell migration assay revealed a ~4 fold increase in the migration of Pt45P1/asTFi cells treated with Dox compared to untreated cells (p<0.001). When cells were co-treated with Dox and inhibitory anti-β1 antibody, migration was inhibited to the levels observed with untreated cells. Migration of Pt45P1/asTFi cells co-treated with Dox and Rb1 was inhibited to the levels observed with Dox/anti-β1 antibody co-treated cells; rabbit isotype control IgG had no effect. Pt45P1 cells, when implanted together with just 100 μg of Rb1, grew significantly smaller tumors that were less vascularized and had ~3.5 fold fewer stromal macrophages compared to the cells implanted together with vehicle (PBS) and/or isotype control IgG (Rb1: avg. wt. 0.835g, 20±9 cells/low power field (LPF); PBS: avg. wt. 1.624g, 73±30 cells/LPF; IgG: avg. wt. 1.665g, 66±47 cells/LPF; p<0.005 Rb1 vs PBS/IgG). Mice in the Pt45P1/Rb1 cohort had ~2 fold decrease in the levels of circulating asTF compared to mice in the Pt45P1/PBS and/or isotype IgG cohorts (0.27 ng/mL vs 0.60/0.54 ng/mL; p<0.001). While Pt45P1 tumors grown in SCID and SCID/TF-low mice were comparable in size, tumor spread was significantly diminished in SCID/TF-low mice (7.74e+8 P/s/mm2 vs 1.09e+9 P/s/mm2, p=0.007); tumors in SCID/TF-low mice also had fewer M2 polarized macrophages (p=0.011) and lower vessel density (4,576µm2/LPF vs 1,834µm2/LPF, p=0.017). In the presence of Rb1, Pt45P1 cells implanted in SCID mice grew tumors significantly smaller compared to IgG control (p=0.005); the effects of Rb1 on primary tumor growth in SCID/low-TF mice were similar to those in SCID mice.We here report that asTF can promote PDAC progression during early and late stages of the disease. asTF-β1 integrin interaction renders PDAC cells more motile, while Rb1 fully inhibits asTF-potentiated PDAC cell migration. Host-derived TF has no impact on the size of primary PDAC tumor, yet it contributes to tumor vascularization and, consequently, metastatic spread. In the presence of Rb1 in nude and/or SCID mice, Pt45P1 cells grow significantly smaller tumors with fewer monocytes and blood vessels that release less asTF in the circulation. Thus, antibody-based targeting of asTF may comprise a novel strategy to stem PDAC progression. DisclosuresNo relevant conflicts of interest to declare.

Dynamic response of ginsenosides in American ginseng to root fungal pathogens

The existence of a complex relationship between soil microbes and ginsenoside contents in their host plants has been reported by previous studies. Here, we analyzed the interaction between the root pathogens and the ginsenoside content in the roots of American ginseng. Two fungal pathogen species were isolated from diseased American ginseng roots, and identified as Fusarium oxysporum (isolate C1) and Fusarium solani (isolate F19) by molecular sequencing analysis. To determine the effect of Fusarium-mediated infection of American ginseng roots, the contents of three ginsenosides, ginsenosides Rb1, ginsenosides Re and ginsenosides Rg1, were monitored over a time course of 120 h post infection using high performance liquid chromatography (HPLC). We found that the level of Rb1 was rapidly upregulated upon fungal infection, whereas the contents of Re or Rg1 were not altered significantly. Furthermore, the presence of Rb1, but not Re or Rg1, significantly inhibited conidium germination of both Fusarium species. Thus, Rb1 is likely the disease-resistance compound produced by American ginseng roots in response to the pathogenic fungi. Our study is the first to report the three ginsenosides with different chemical structure respond differently to soilborne pathogen infection.

Ginsenoside rb1 and rg3 attenuate glucocorticoid-induced neurotoxicity.

Glucocorticoid (GC) hormones, increased in response to stress, can cause neuronal loss. We tested the effect of GC hormone on cell viability of neural SHSY-5Y cells and protective effects of ginsenoside Rb1 and Rg3 on the action of GC. We treated SHSY-5Y cells with increasing concentrations of synthetic GC dexamethasone (DEX; 10, 25, 50, and 100 nM) for 24 and 48 h, and then determined cell viability by using MTT assay. We then treated SHSY-5Y cells with DEX (100 nM) with or without the ginsenosides to examine their preventive effects on the cytotoxicity. To explore the underlying molecular mechanisms, we measured mRNA expression of bax and bcl-2 by using reverse transcriptase real-time PCR. SHSY-5Y cells treated with DEX significantly reduced cell viability as compared with control cells. In the presence of Rb1 or Rg3, DEX-induced cytotoxicity was effectively blocked. DEX considerably increased pro-apoptotic bax mRNA expression as compared with control cells. However, Rb1 and Rg3 completely blocked DEX-mediated up-regulation of bax expression. DEX significantly increased neuronal death in organotypic hippocampal slice cultures of rat brain with enhanced generation of ROS, which was effectively inhibited by ginsenoside Rb1 and Rg3. This suggests a potential role of the ginsenosides to target GC action in the brain.

Effects of Homocysteine and Ginsenoside Rb1 on Endothelial Proliferation and Superoxide Anion Production

Homocysteine (Hcy) is an independent risk factor for cardiovascular disease by its multiple effects on vascular cells and throbmosis factors, which may be involved in oxidative stress mechansims. Ginsenoside Rb1, a constituent of ginseng, bears various beneficial effects on the cardiovascular system. In the present study, we investigated the effect of Hcy on endothelial proliferation and a protective effect of ginsenoside Rb1 on the action of Hcy. We initially incubated a mouse lymph node endothelial cell line (SVEC4-10) with increasing concentrations of Hcy or for different time periods and then assessed cell proliferation by using [(3)H]-thymidine incorporation. We then incubated SVEC4-10 cells with Hcy (50 microM) for 24 h with or without Rb1 (10 microM) to examine its inhibitory effect on the proliferation. These experiments were repeated in human umbilical vein endothelial cells (HUVECs). To explore the underlying molecular mechanisms, we measured superoxide anion, a reactive oxygen species (ROS), by using dihydroethidium (DHE) staining. SVEC4-10 cells treated with Hcy (50, 100, and 200 microM) for 24 h significantly reduced cell proliferation by 43%, 42%, and 40%, respectively, as compared with control cells (P < 0.01). SVEC4-10 cells treated with Hcy (50 microM) for 12 and 24 h showed a significant reduction of cell proliferation (P < 0.05). In HUVECs, Hcy (50 microM) significantly reduced cell proliferation by 55% as compared with control cells (P < 0.05). In the presence of Rb1, Hcy-induced inhibition of cell proliferation was effectively blocked in both SVEC4-10 and HUVECs. Furthermore, Hcy (50 microM) significantly increased superoxide anion production by 23% in SVEC4-10 as compared with control cells (P < 0.05). However, in the presence of Rb1, Hcy increased superoxide anion production by only 8%, showing that RB1 almost completely blocked the effect of Hcy. Hcy significantly inhibits endothelial proliferation with increased production of superoxide anion, which is effectively blocked by ginsenoside Rb1. This study provides some new aspects of Hcy-induced endothelial dysfunction, and suggests a potential role of Rb1 to block Hcy action, which may have clinical applications.

Transfection of an inducible p16/CDKN2A construct mediates reversible growth inhibition and G1 arrest in the AtT20 pituitary tumor cell line.

Recent studies have shown that methylation of the CpG island within the p16/CDKN2A gene is associated with an absence of p16 protein in human pituitary tumors. However, the effect of restoration of p16 protein expression in this tumor type has not been investigated. In the absence of an available human pituitary cell line we first assessed the suitability of the mouse corticotroph cell line AtT20 as a model system. Initial experiments showed that the p16/CDKN2A gene was not expressed, whereas a transcript for RB1 was detected as assessed by RT-PCR. Further studies showed the p16/CDKN2A gene to be homozygously deleted. The absence of p16/CDKN2A and presence of RB1, the down-stream effector of p16-mediated cell cycle arrest confirmed the suitability of the AtT20 cell line as a model system. Stable transfectants were generated in which p16/CDKN2A is regulated by an inducible promoter. The regulatory effects of p16/CDKN2A expression on cell proliferation were assessed and complemented by fluorescence-activated cell sorting (FACS) analysis of cell cycle profile. Induced expression of p16/CDKN2A resulted in a profound inhibition of cell growth and G1 arrest (80-82%). Western blot analysis showed concomitant expression of p16 protein in arrested cells and a shift in the phosphorylation status of pRB toward its hypophosphorylated form. To further confirm that expression of p16/CDKN2A mimicked its in vivo role, reversibility was assessed using alternate cycles in the presence and absence of inducer (isopropyl-1-thio-beta-D-galactopyranoside). Over three cycles the absence of induced expression of p16/CDKN2A resulted in release from G1 arrest. These results show that, in a pituitary cell line model, restoration of p16 expression is indeed sufficient to arrest cells in G1 and inhibit cell proliferation and is reversible. Thus restoration of p16 expression through novel strategies, including gene therapy or demethylating agents, may offer successful therapeutic intervention in human forms of this disease.

Transfection of an Inducible p16/CDKN2A Construct Mediates Reversible Growth Inhibition and G1 Arrest in the AtT20 Pituitary Tumor Cell Line

Recent studies have shown that methylation of the CpG island within the p16/CDKN2A gene is associated with an absence of p16 protein in human pituitary tumors. However, the effect of restoration of p16 protein expression in this tumor type has not been investigated. In the absence of an available human pituitary cell line we first assessed the suitability of the mouse corticotroph cell line AtT20 as a model system. Initial experiments showed that the p16/CDKN2A gene was not expressed, whereas a transcript for RB1 was detected as assessed by RT-PCR. Further studies showed the p16/CDKN2A gene to be homozygously deleted. The absence of p16/CDKN2A and presence of RB1, the downstream effector of p16-mediated cell cycle arrest confirmed the suitability of the AtT20 cell line as a model system. Stable transfectants were generated in which p16/CDKN2A is regulated by an inducible promoter. The regulatory effects of p16/CDKN2A expression on cell proliferation were assessed and complemented by fluorescence-activated cell sorting (FACS) analysis of cell cycle profile. Induced expression of p16/CDKN2A resulted in a profound inhibition of cell growth and G1 arrest (80–82%). Western blot analysis showed concomitant expression of p16 protein in arrested cells and a shift in the phosphorylation status of pRB toward its hypophosphorylated form. To further confirm that expression of p16/CDKN2A mimicked its in vivo role, reversibility was assessed using alternate cycles in the presence and absence of inducer (isopropyl-1-thio-β-d-galactopyranoside). Over three cycles the absence of induced expression of p16/CDKN2A resulted in release from G1 arrest. These results show that, in a pituitary cell line model, restoration of p16 expression is indeed sufficient to arrest cells in G1 and inhibit cell proliferation and is reversible. Thus restoration of p16 expression through novel strategies, including gene therapy or demethylating agents, may offer successful therapeutic intervention in human forms of this disease.