scholarly journals Knockdown of the oncogene lncRNA NEAT1 restores the availability of miR-34c and improves the sensitivity to cisplatin in osteosarcoma

2018 ◽  
Vol 38 (3) ◽  
Author(s):  
Yuliang Hu ◽  
Qiuyong Yang ◽  
Long Wang ◽  
Shuo Wang ◽  
Fei Sun ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Tumor Growth ◽  
Cyclin D1 ◽  
Clinical Significance ◽  
Cell Lines ◽  
Target Genes ◽  
Tumor Regression ◽  
G1 Arrest ◽  
Ki 67

Aberrant expressions of long non-coding RNAs (lncRNAs) are the culprits of carcinogenesis via regulating the tumor suppressor or oncogene. LncRNA nuclear enriched abundant transcript 1 (NEAT1) has been identified to be an oncogene to promote tumor growth and metastasis of many cancers. However, the clinical significance and function of NEAT1 in osteosarcoma (OS) remain to be discovered. We here collected OS tissues (n=40) and adjacent non-tumor tissues (n=20) to determine the expression of NEAT1 and its clinical significance. NEAT1 was overexpressed in OS tissues, which positively correlated with tumor size, Enneking stage, and distant metastasis of OS patients. The elevated level of NEAT1 was confirmed in OS cell lines including MG63 and HOS in vitro. Knockdown of NEAT1 by two siRNAs induced impaired cell vitalities, promoted the apoptosis, and G0/G1 arrest in two cell lines, which was associated with inhibited anti-apoptosis signals BCL-2 pathway and cell cycle-related cyclin D1 (CCND1) signals. Moreover, the tumor suppressor miR-34c was negatively regulated and inhibited by NEAT1 in OS. Suppression of miR-34c could up-regulate the expressions of its target genes BCL-2 and CCND1 to antagonize the effects of NEAT1 knockdown. Furthermore, overexpressed NEAT1 reduced the sensitivity of cisplatin (DDP) and inhibited DDP-induced apoptosis and cell cycle arrest via miR-34c. The results in vivo also confirmed that knockdown of NEAT1 sensitized the OS cells to DPP-induced tumor regression, delayed the tumor growth with reduced levels of Ki-67, BCL-2, and cyclin D1 signals, suggesting that NEAT1 is an oncogene and chemotherapy resistant factor in OS.

Depsipeptide in the Treatment of Relapsed and Refractory Multiple Myeloma (MM): A Prospective Evaluation of the Cell Cycle.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1497-1497
Author(s):  
Zoe Goldberg* ◽  
Scott Ely ◽  
Selina Chen-Kiang ◽  
Martha Chesi ◽  
Peter L. Bergsagel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Phase Ii ◽  
Caspase 3 ◽  
Gene Array ◽  
Cyclin D3 ◽  
Proliferation Index ◽  
G1 Arrest

Abstract Background: Dysregulation of the cell cycle and apoptosis are two critical events in the pathophysiology of MM. This notion is supported by: 1)A high tumor burden is often present despite a low rate of tumor cell proliferation. 2)G1 arrest is common in MM cells while normal plasma cells are permanently withdrawn from cell cycle. 3) Cyclin D1 is often overexpressed without a defined genetic substrate. Herein, we show that cell cycle evaluation in vivo is feasible and that the histone-deacetylase inhibitor depsipeptide might be effective in selected patients with MM. Patients and Methods: In vitro studies were performed in 12 human MM cell lines with defined cytogenetic abnormalities. The IC50 for depsipeptide was determined by evaluation of apoptosis by standard methods. In vivo studies where done as correlates in a phase II protocol. These include: Immunohistochemistry (IHC) for co-expression of CD138/Ki-67 as a proliferation index (PCPI), cyclin D1, D3, caspase 3 cleavege, CD31 and bcl-2 before treatment and at 24 hrs and 30 days after treatment. Gene array studies are being performed on selected patients at those timepoints. To date, four stage III patients (PTS) with relapsed MM with four or fewer prior lines of therapy have been treated with one to three cycles of depsipeptide at a dose of 13mg/m2,as a 4-hour infusion on days 1, 8, and 15, repeated every 28 days. Mean age was 63 years (range, 56 to 72). KPS of >80%. Mean albumin was 3.5, (range, 3.2 to 4), mean LDH was 243 (range, 179 to 315). Results: 1)Depsipeptide induces apoptosis in several MM cell lines. All lines were susceptible to depsipeptide, however, differential sensitivities were noted. Three cell lines (ie U266) that contained 11q13 translocation (cyclin D1 overexpression) were the most sensitive with IC50s at least 2 fold lower than other lines. 2) Cell cycle changes are induced by depsipeptide: In 2/4 PTS, a significant increase of the PCPI was seen, whereas a marked reduction in the PCPI in a patient with cyclin D3 overexpression (27% to 16%) was also noted. One patient had an increase of cyclin D1 post treatment. No changes where seen in bcl-2, CD-31, or cleaved caspase-3 expression. 3) Depsipeptide is safe in a limited cohort of MM PTS: Grade 2 fatigue and anorexia were the most common toxicities. Mild thrombocytopenia (mean of 67) did not require transfusions. One patient had stable disease after 3 cycles of treatment, one patient had progression of disease after 3 cycles, one patient progressed after the 1st cycle, and one patient is too early for evaluation. Conclusions: 1)Patients with 11q13 translocation should be a target for treatment with depsipeptide. 2)Depsipeptide given on this schedule is safe and can stabilize tumor-mass in PTS with otherwise progressive relapsed and refractory disease.3) Evidence of cell cycle modulation can be seen during treatment with depsipeptide. No profound changes in apoptosis is evident.4)Further studies may help to understand the mechanism of transcriptional regulation by depsipeptide and will help design rational therapy and combinations. This study continues to accrue patients as part of New York Phase II Consortium. Supported by NCI grant (SAIC1N01-CO-12400-02) and a SCOR for Myeloma grant from the Leukemia and Lymphoma Society of America.

Effect of microRNA-206 on G1 arrest by inhibition of CDK4, cyclin C, and cyclin D in melanoma cells.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e20047-e20047 ◽  
Author(s):  
Robert W Georgantas ◽  
Katie Streicher ◽  
Xiaobing Luo ◽  
Wei Zhu ◽  
Zheng Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Normal Skin ◽  
G1 Arrest ◽  
Mrna Targets ◽  
Cyclin C ◽  
Skin Biopsies ◽  
Melanoma Cell Lines

e20047 Background: MiR-206 has been implicated in a large number of cancers. However, its role in tumor biology is unknown and its biological function has yet to be fully characterized. To examine the role of miR-206 in cancer, we examined the expression of miR-206 in melanoma and identified potential target transcriptss that could be important for the progression of this disease. Methods: Using quantitative RT-PCR we compared expression of 364 microRNAs in melanoma skin biopsies skin from normal donors, melanoma cell lines, and normal melanocytes. The effects of miR-206 on cell growth, apoptosis, and cellular migration/invasion were determined using in vitro assays comparing melanoma cell lines to normal melanocytes. Putative mRNA targets of miR-206 were bioinfomatically identified, and empirically tested by luciferase-3’UTR reporter assays. The effect of miR-206 on the cell cycle of melanoma cells was assayed by flow cytometry. Results: Expression profiling of microRNAs in melanoma lesional skin biopsies compared to normal donor skin biopsies revealed numerous differentially regulated miRs. One such microRNA, miR-206, was significantly highly down-regulated in melanoma biopsies (-75.4-fold, p=1.7x10-4) compared to normal skin and normal melanocytes. Functional analysis showed that miR-206 substantially reduced growth and migration/invasion of multiple melanoma cell lines. Bioinformatic analysis identified the cell cycle genes CDK2, CDK4, Cyclin C, and Cyclin D1 as strong candidate mRNA targets of miR-206. Luciferase reporter gene assays revealed that miR-206 inhibits translation of CDK4, Cyclin D1, and Cyclin C. Consistent with this inhibition of CDK4 and Cyclin D1, miR-206 transfection induced robust G1 arrest in multiple melanoma cell lines. Conclusions: MiR-206 expression was decreased in melanoma tissue and cell lines compared to normal skin and melanocytes, respectively. Inhibition of Cyclin C, Cyclin D1 and CDK4 by miR-206 highlights its role in regulating cell cycle progression, a key aspect of melanoma progression. These observations support miR-206 as a potential tumor suppressor in melanoma, and possibly other cancers.

scholarly journals Integrative transcriptomics analysis for uterine leiomyosarcoma identifies aberrant activation of cell cycle-dependent kinases and their potential therapeutic significance.

2022 ◽  
Author(s):  
Kosuke Yoshida ◽  
Akira Yokoi ◽  
Tomofumi Yamamoto ◽  
Yusuke Hayashi ◽  
Jun Nakayama ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Ingenuity Pathway Analysis ◽  
Pathway Analysis ◽  
Transcriptome Analysis ◽  
Target Genes ◽  
Tumor Regression ◽  
Uterine Leiomyosarcoma ◽  
Anti Cancer ◽  
Bi 2536

Purpose: Uterine leiomyosarcoma is among the most aggressive gynecological malignancies. No effective treatment strategies have been established. This study aimed to identify novel therapeutic targets for uterine leiomyosarcoma based on transcriptome analysis and assess the preclinical efficacy of novel drug candidates. Experimental Design: Transcriptome analysis was carried out using fresh-frozen samples of six uterine leiomyosarcomas and three myomas. The Ingenuity Pathway Analysis was then used to identify potential therapeutic target genes for uterine leiomyosarcoma. Moreover, our results were validated using three independent datasets, including 40 uterine leiomyosarcomas. Then, the inhibitory effects of several selective inhibitors for the candidate genes were examined using the SK-UT-1, SK-LMS-1, and SKN cell lines. Results: We identified 512 considerably dysregulated genes in uterine leiomyosarcoma compared with myoma. The Ingenuity Pathway Analysis showed that the function of several genes, including CHEK1 and PLK1, were predicted to be activated in uterine leiomyosarcoma. Through an in vitro drug screening, PLK1 or CHEK1 inhibitors (BI 2536 or prexasertib) were found to exert a superior anti-cancer effect against cell lines at low nanomolar concentrations and induced cell cycle arrest. In SK-UT-1 tumor-bearing mice, BI 2536 monotherapy demonstrated a marked tumor regression. Moreover, the prexasertib and cisplatin combination therapy also reduced tumorigenicity and prolonged survival. Conclusion: We identified the upregulated expression of PLK1 and CHEK1; their kinase activity was considered to be activated in uterine leiomyosarcoma. BI 2536 and prexasertib demonstrate a significant anti-cancer effect; thus, cell cycle-related kinases may represent a promising therapeutic strategy for treating uterine leiomyosarcoma.

scholarly journals The RASSF1A Tumor Suppressor Blocks Cell Cycle Progression and Inhibits Cyclin D1 Accumulation

2002 ◽  
Vol 22 (12) ◽  
pp. 4309-4318 ◽  
Author(s):  
Latha Shivakumar ◽  
John Minna ◽  
Toshiyuki Sakamaki ◽  
Richard Pestell ◽  
Michael A. White
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Phase Cell ◽  
Cycle Progression ◽  
Cycle Arrest

ABSTRACT The RASSF1A locus at 3p21.3 is epigenetically inactivated at high frequency in a variety of solid tumors. Expression of RASSF1A is sufficient to revert the tumorigenicity of human cancer cell lines. We show here that RASSF1A can induce cell cycle arrest by engaging the Rb family cell cycle checkpoint. RASSF1A inhibits accumulation of native cyclin D1, and the RASSF1A-induced cell cycle arrest can be relieved by ectopic expression of cyclin D1 or of other downstream activators of the G1/S-phase transition (cyclin A and E7). Regulation of cyclin D1 is responsive to native RASSF1A activity, because RNA interference-mediated downregulation of endogenous RASSF1A expression in human epithelial cells results in abnormal accumulation of cyclin D1 protein. Inhibition of cyclin D1 by RASSF1A occurs posttranscriptionally and is likely at the level of translational control. Rare alleles of RASSF1A, isolated from tumor cell lines, encode proteins that fail to block cyclin D1 accumulation and cell cycle progression. These results strongly suggest that RASSF1A is an important human tumor suppressor protein acting at the level of G1/S-phase cell cycle progression.

scholarly journals m⁶A Demethylase ALKBH5 Promotes Tumor Cell Proliferation by Destabilizing IGF2BPs Target Genes and Worsens the Prognosis of Patients with Non-Small Cell Lung Cancer

2021 ◽  
Author(s):  
Kazuo Tsuchiya ◽  
Katsuhiro Yoshimura ◽  
Yuji Iwashita ◽  
Yusuke Inoue ◽  
Tsutomu Ohta ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Cell ◽  
Target Genes ◽  
Cancer Cell Lines ◽  
G1 Arrest ◽  
Lung Cancer Cell ◽  
Proliferation Ability

Abstract Background: The modification of N6-methyladenosine (m6A) in RNA and its eraser ALKBH5, an m6A demethylase, play important roles across various steps of human carcinogenesis. However, the involvement of ALKBH5 in non-smallcell lung cancer (NSCLC) development remains to be completely elucidated.Methods: The current study investigated the involvement of ALKBH5 in NSCLC development using immunostaining of clinical NSCLC specimens as well as cancer-related cellular functions (cell proliferation, migration ability, cell cycle, and apoptosis) in ALKBH5-knockdown lung cancer cell lines. Moreover, a microarray was utilized to comprehensively analyze mRNA and m6A in ALKBH5-knockdown cells. m6A target genes were identified using the methylated RNA immunoprecipitation (MeRIP) assay with m6A antibody. Furthermore, mRNA stability and protein expression owing to m6A modification (the target genes) were examined.Results: Clinicopathological analysis revealed that increased ALKBH5 expression was an independent prognostic factor associated with unfavorable overall survival in NSCLC (hazard ratios, 1.468; 95% confidence interval, 1.039–2.073). In vitro study revealed that ALKBH5 knockdown suppressed cell proliferation ability of PC9 and A549 cells as well as promoted G1 arrest and increased the number of apoptotic cells. Furthermore, ALKBH5 overexpression increased the cell proliferation ability of the immortalized cell lines BEAS2B and HEK293. Comprehensive analysis of microarray and MeRIP quantitative-polymerase chain reaction revealed that 3′ untranslated regions (3′ UTRs) of CDKN1A, TIMP3, E2F1, and CCNG2 mRNA were potential targets of ALKBH5. Depending on the lung cancer cell lines, increased expression of CDKN1A or TIMP3 and decreased cell proliferation were observed by ALKBH5 knockdown.These alterations were offset by a double knockdown of both ALKBH5 and one of the IGF2BPs. The decline of mRNAs was, at least partly, owing to the destabilization of these mRNAs by one of the IGF2BPs.Conclusions:Upregulation of ALKBH5 in NSCLC reduces m6A modifications on the 3′ UTR of specific genes. Loss of m6A causes a decrease in opportunity for recognition by IGF2BPs and destabilizes the target transcript, such as CDKN1A (p21) and TIMP3. Downregulation of CDKN1A (p21) and TIMP3 induces cell cycle alteration and inhibits apoptosis. The ALKBH5–IGF2BPs axis promotes cell proliferation and tumorigenicity, which in turn causes the unfavorable prognosis of NSCLC.

Inhibition of CKS1B Expression Affects Myeloma Cell Growth In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 370-370
Author(s):  
Bangzheng Chen ◽  
Ling Liu ◽  
Kristin L. Mitchell ◽  
Xiasong Wu ◽  
Simona Colla ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Growth ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Proteasomal Degradation ◽  
Myeloma Cell ◽  
High Expression ◽  
G1 Arrest ◽  
Shrna Expression

Abstract CKS1B is the regulatory component of the SCF-Skp2 ubiquitin ligase that ubiquinates the cyclin-dependent kinase inhibitor p27kip1 targeting it for proteasomal degradation. Overexpression of CKS1B is associated with poor prognosis in malignancies such as oral squamous cell carcinomas, colorectal carcinoma, breast cancer, prostate cancer, and others. Recently, high expression levels of CKS1B were associated with an aggressive course of Multiple Myeloma (Shaughnessy J. Hematology. 2005;10 Suppl 1:117). We investigated whether inhibition of CKS1B in myeloma cell lines with multiple copies of 1q21 and high expression of CKS1B affects in vivo tumor growth. Myeloma cell lines were transduced with lentivirus conditionally expressing (tet on) CKS1B shRNA and lentivirus constitutively expressing luciferase (for in vivo experiments), or with lentivirus constitutively expressing CKS1B shRNA (for in vitro work). Cells with inducible expression were injected into the human bones of SCID-hu mice, and, once tumors established, expression was induced with doxycycline in the drinking water. Changes in tumor sizes were followed by weekly luminescence imaging. Cells with constitutive shRNA expression were used to determine effects on cell cycle by propidium iodide flow cytometry. Effects of shRNA expression on cytoplasmic and nuclear CKS1B and p27kip1 levels were determined by western blot analysis. Induction of CKS1B shRNA expression in JJN3 and OCI-MY5 cells in SCID-hu mice inhibited tumor growth by 57% and 81% within one week, compared to non-targeting construct. As expected, viable cells recovered from these tumors had low levels of CKS1B and higher levels of p27kip1 proteins. Similar to observations in the MM cell lines JJN3 and OCI-MY5 (see Abstract by Zhan and colleagues), constitutive expression of shRNA in CAG and XG1 cells effectively reduced cytoplasmic and nuclear CKS1B protein levels. This was associated with increased levels of p27kip1 protein, marked (51%) increases of cells in the G1 phase of the cell cycle, and reduced S and G2 (33% and 52%, respectively), compatible with a G1 arrest, and a 76% increase in the number of sub G1 (apoptotic) cells. In contrast to these observations, cells constitutively over-expressing p27kip1 had no change in G1, 50% fewer cells in S, and a 150% increase in G2, indicating G1 and G2 arrests, with no change in the sub G1 fraction. Interestingly, cells over-expressing p27kip1 had lower levels of CKS1B proteins, compatible with the reported proteasomal degradation of the molecule during the later phases of the cell cycle (Hattori T et al, Genes Cells.2003;8:889). These observations suggest that the poor prognosis associated with 1q21 amplifications and high CKS1B expression in myeloma reflects an elevated proliferative state of the tumor cells that results in rapid regrowth of minimal residual disease.

scholarly journals The Novel CDK4/6-Inhibitor Abemaciclib Induces Early G1-Arrest in MCL Cell Lines, Sensitizes Cells to Cytarabine Treatment and Is Additive with Ibrutinib

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5124-5124
Author(s):  
Luca Fischer ◽  
Andrea Schnaiter ◽  
Bianca Freysoldt ◽  
Markus Irger ◽  
Yvonne Zimmermann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
S Phase ◽  
G1 Phase ◽  
G1 Arrest ◽  
The Novel ◽  
Sensitive Cell ◽  
Cycle Arrest ◽  
Arac Treatment

Abstract Introduction: Mantle cell lymphoma (MCL) is characterized by t(11;14) resulting in a constitutive cyclin D1 overexpression. The cyclin D1-CDK4/6 complex inactivates Rb through phosphorylation, leading to G1/S-phase transition. Therefore, inhibition of CDK4/6 is an efficient and rational approach to overcome cell cycle dysregulation in MCL. We evaluated the efficiency of the novel CDK4/6 inhibitor abemaciclib in various MCL cell lines and in primary MCL cells in combination with cytarabine (AraC) and ibrutinib. Material & Methods: MCL cell lines (Granta 519, JeKo-1, Maver-1, Mino) and primary MCL cells were exposed to abemaciclib alone and combined with AraC or ibrutinib. Cells were pretreated with abemaciclib and exposed to AraC or ibrutinib with or without consecutive wash-out of the CDK4/6 inhibitor. Proliferation and viability were measured by tryptan blue staining and Cell Titer Glo assay. Flow cytometry was used for cell-cycle (PI-staining) and apoptosis analysis (Annexin V PE/7AAD-staining). Western Blot analysis showed protein expression and phosphorylation status of various downstream proteins. Results: Abemaciclib inhibited cell proliferation by induction of early G1-arrest. Western Blot analysis revealed reduced phosphorylation of Rb on serine 795 without changes in CDK 4 and cyclin D1 expression, in line with reversible cell cycle arrest. IC50-values of sensitive cell lines (JeKo-1, Maver-1, Mino) were <30 nM after 72 h. We observed an almost complete and reversible G1-arrest in all sensitive cell lines by FACS analysis (JeKo-1: G1-phase +51,7 %; S/G2-phase -51,7 % at 31,25 nM after 24 h; G1-phase +35,4 %; S/G2-phase -34,8 % after 72 h), whereas cell viability was not reduced. Wash-out of abemaciclib after 24 h resulted in synchronized S-phase entry in all sensitive cell lines (e.g. Mino: G1-phase -20,4 %; S-phase +30,5 %). The sequential combination of abemaciclib followed by AraC showed strong synergy in Mino cells (CI=0,22 for 31,25 nM abemaciclib and 3,33 µM cytarabine). In contrast, simultaneous exposure to abemaciclib had a protective effect against AraC treatment in all sensitive cell lines, due to an ongoing G1-arrest (Mino: CI=-0,19 for 31,25 nM abemaciclib and 3,33 µM AraC). In primary MCL cells, 31,25 nM of abemaciclib had no impact on cell death. Moreover, no sensitization to AraC was observed as all cells were resting in G0-phase. The combination of abemaciclib induced G1 arrest and ibrutinib had additive or synergistic effects in sensitive cell lines (JeKo-1, Mino and Maver). Conclusion: The novel CDK4/6 inhibitor abemaciclib causes reversible G1 cell cycle arrest without loss of viability at low nanomolar doses. Rationale drug combinations exploiting the sequential effect may achieve major benefits, but drug interactions are complex: Pretreatment with abemaciclib sensitizes MCL cell line cells to AraC whereas simultaneous application protects them from AraC treatment. Further analyses explore the interaction with other targeted approaches (inhibitors of the B-cell receptor pathway) to better understand the underlying molecular mechanisms. Disclosures No relevant conflicts of interest to declare.

scholarly journals DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

1994 ◽  
Vol 125 (3) ◽  
pp. 625-638 ◽  
Author(s):  
J Lukas ◽  
H Müller ◽  
J Bartkova ◽  
D Spitkovsky ◽  
A A Kjerulff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Complex Formation ◽  
Cyclin D1 ◽  
Cell Lines ◽  
T Antigen ◽  
Regulatory Function ◽  
Retinoblastoma Gene ◽  
Checkpoint Control ◽  
Wild Type ◽  
In Cells

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.

A novel PLK1 inhibitor onvansertib effectively sensitizes MYC-driven medulloblastoma to radiotherapy

2021 ◽  
Author(s):  
Dong Wang ◽  
Bethany Veo ◽  
Angela Pierce ◽  
Susan Fosmire ◽  
Krishna Madhavan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Tumor Growth ◽  
Radiation Treatment ◽  
Tumor Regression ◽  
Tumor Growth Inhibition ◽  
Cell Cycle Distribution ◽  
Cell Renewal ◽  
Group 3

Abstract Background Group 3 medulloblastoma (MB) is often accompanied by MYC amplification. PLK1 is an oncogenic kinase that controls cell cycle and proliferation and has been preclinically validated as a cancer therapeutic target. Onvansertib (PCM-075) is a novel, orally available PLK1 inhibitor, which shows tumor growth inhibition in various types of cancer. We aim to explore the effect of onvansertib on MYC-driven medulloblastoma as a monotherapy or in combination with radiation. Methods Crisper-Cas9 screen was used to discover essential genes for MB tumor growth. Microarray and immunohistochemistry on pediatric patient samples were performed to examine the expression of PLK1. The effect of onvansertib in vitro was measure by cell viability, colony-forming assays, extreme limiting dilution assay and RNA-Seq. ALDH activity, cell-cycle distribution and apoptosis were analyzed by flow cytometry. DNA damage was assessed by immunofluorescence staining. Medulloblastoma xenografts were generated to explore the monotherapy or radio-sensitizing effect. Results PLK1 is overexpressed in Group 3 MB. The IC50 concentrations of onvansertib in Group 3 MB cell lines were in a low nanomolar range. Onvansertib reduced colony formation, cell proliferation, stem cell renewal and induced G2/M arrest in vitro. Moreover, onvansertib in combination with radiation increased DNA damage and apoptosis compare with radiation treatment alone. The combination radiotherapy resulted in marked tumor regression in xenografts. Conclusions These findings demonstrate the efficacy of a novel PLK1 inhibitor onvansertib in vitro and in xenografts of Group 3 MB, which suggests onvansertib is an effective strategy as monotherapy or in combination with radiotherapy in MB.

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