Chemosensitisation by poly(ADP-ribose) polymerase-1 (PARP-1) inhitor 5-aminoisoquinoline (5-AIQ) on various melanoma cell lines

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 12019-12019 ◽  
Author(s):  
S. Radulovic ◽  
S. Bjelogrlic ◽  
Z. Todorovic ◽  
M. Prostran
Keyword(s):  
Cell Cycle ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Cell Cycle Progression ◽  
Single Agent ◽  
S Phase ◽  
G1 Arrest ◽  
Parp 1 ◽  
Melanoma Cell Lines

12019 Background: PARP-1 facilitates DNA strand brakes repair and PARP inhibitors were investigated as enhancers of chemoradiotherapy. We investigated whether 5-AIQ potentates the effect of doxorubicin (DOXO), cisplatin (CDDP) and paclitaxel (Ptx) on human (slow-growing) FemX and murine (fast-growing) B16 melanoma cell lines. Methods: Twenty-four hours after cells were seeded in 96 well plates, cytotoxic drugs and 5-AIQ were added to cell medium. For evaluation of single-agent activity, drugs were applied in concentration ranges as follows: CDDP (0.3–30 μM), DOXO (0.1–3 μM), Ptx (1–100 ηM), 5-AIQ (1–100 μM). 5-AIQ (3μM) was combined with CDDP (0.1, 0.3, 1 μM), DOXO (10, 3, 100 ηM), or Ptx (1, 3, 10 ηM). Incubation lasted for 72 hrs when SRB assay was utilized to determine individual and combine activity (interactions calculated with isobole method). For cell cycle analysis B16 cells were seeded on 6 well plates and treated with each drug alone and combinations, using the same concentrations as those for investigation of combine cytotoxic activity. Cell cycle was determined after 72 hrs, on FACS Calibur with propidium iodide dye. Results: 5-AIQ induced minimal changes in cell viability and cell cycle progression on both cell lines, compared to non-treated control. CDDP revealed high activity against FemX (IC50 = 2.85 μM) and B16 cells (IC50 = 8.84 μM), and G0/G1 arrest. In B16 cells 5-AIQ multiply enhanced CDDP’s activity with strong synergistic interaction and cells slightly driven to S phase. Synergism was also detected on B16 cells treated with combination of DOXO (IC50 = 0.2 μM on B16 and 0.89 μM on FemX) and 5-AIQ when DOXO was applied in low concentrations (10 and 30 ηM), while 5-AIQ did not interfere with cell cycle changes. Cytotoxicity of Ptx (IC50 = 6.16 ηM on B16 and <1 ηM on FemX) was stimulated only at higher concentrations. 5-AIQ stimulated G0/G1 and S phase arrest on B16 cells with Ptx of 3 and 10 ηM, respectively. In FemX cells, most of the interactions of 5-AIQ with CDDP, DOXO, and Ptx revealed as antagonistic. Conclusions: PARP-1 inhibitor 5-AIQ enhances cytotoxic activity of both DNA damaging and agents with different mechanism of action, but the effect varies between cell lines with different proliferation rate. No significant financial relationships to disclose.

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.

Effect of celastrol on temozolomide cytotoxicity in melanoma cells and inhibition of NF-kB signaling

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 9076-9076
Author(s):  
M. Chen ◽  
I. Osman ◽  
S. J. Orlow
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Natural Products ◽  
Natural Product ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Single Agent ◽  
Melanoma Cells ◽  
Cell Killing ◽  
Melanoma Cell Lines

9076 Background: Temozolomide (TMZ) exhibits clinical activity in the treatment of melanoma and glioblastoma, but response rates are low. Identification of agents that improve TMZ's efficacy and overcome resistance is of great interest. In this study, we have identified celastrol as a natural product that significantly enhances TMZ-induced cytotoxicity by testing a library of drugs and natural products for cytotoxic activity against glioma and melanoma cell lines and have examined its mechanism of action in melanoma cells. Methods: A preliminary screening of a library of 2000 drugs and natural products was performed and a short list of drugs was identified as able to enhance TMZ-induced cell killing in TMZ-resistant cancer cell lines. The effects of these compounds were further confirmed in five melanoma cell lines. A cell proliferation assay was used to compare growth inhibitory effects of single agent TMZ versus combination treatments. Synergy in inhibiting cell proliferation was assessed using combination-index methods. The expression of NF-kB, IkB, MAPK, and PARP were examined using Western blot analysis. The effect of treatments on the cell cycle was examined by flow cytometry. The localization of NF-kB in melanoma cells was evaluated through immunofluorescence microscopy. Results: Combining celastrol and TMZ synergistically inhibited cell proliferation, enhanced cell cycle arrest, and increased apoptosis in a series of melanoma cell lines, compared to treatment with TMZ alone. We further found that celastrol inhibited proteasome activity, TNF-α induced IkB phosphorylation and NF-kB translocation to the nucleus. Inhibition of NF-kB with siRNA mimicked the ability of celastrol to sensitize melanoma cells to TMZ-induced cell killing, suggesting inhibition of NF-kB was indeed involved in TMZ/celastrol-induced cytotoxicity. Furthermore, combination treatment induced phosphorylation of JNK. Conclusions: Our data suggest that combined use of TMZ with celastrol, a natural product derived from a vine extract that has been used orally in Chinese medicine for over a thousand years, may enhance the chemotherapeutic efficacy of TMZ in melanoma. No significant financial relationships to disclose.

The Inhibition of Checkpoint Kinase 1 As a Promising Strategy to Increase the Effectiveness of Different Treatments in Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2478-2478
Author(s):  
Andrea Ghelli Luserna Di Rora ◽  
Ilaria Iacobucci ◽  
Enrica Imbrogno ◽  
Enrico Derenzini ◽  
Anna Ferrari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Viability ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Propidium Iodide ◽  
Lymphoblastic Leukemia ◽  
Single Agent ◽  
Dna Damages ◽  
Checkpoint Kinase

Abstract Nowadays the effectiveness of the treatments for adult Acute Lymphoblastic Leukemia (ALL) patients is still inadequate and frequently many patients after years of response to treatments develop relapses. Thus there is a need to find novel targets for specific therapies and to maximize the effect of the actual treatments. Recently different Checkpoint Kinase (Chk)1/Chk2 inhibitors has been assessed for the treatment of different type of cancers but only few studies have been performed on hematological diseases. We evaluated the effectiveness of the Chk1 inhibitor, LY2606368, as single agent and in combination with tyrosine kinase inhibitors (imatinib and dasatinib) or with the purine nucleoside antimetabolite clofarabine in B-/T- acute lymphoblastic leukemia (ALL) cell lines and in primary blasts. Human B (BV-173, SUPB-15, NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines were incubated with increasing concentrations of drug (1-100 nM) for 24 and 48 hours and the reduction of the cell viability was evaluated using WST-1 reagent. LY2606368 deeply reduced the cell viability in a dose and time dependent manner in all the cell lines, with the BV-173 (6.33 nM IC50 24hrs) and RPMI-8402 (8.07 nM IC50 24hrs) being the most sensitive while SUP-B15 (61.4 nM IC50 24hrs) and REH (96.7 nM IC50 24hrs) being the less sensitive cell lines. Moreover the sensitivity to the compound was no correlated with the different sub-type of ALL or with the mutational status of p53, which is a marker of the functionality of the G1/S checkpoint. The cytotoxic activity was confirmed by the significant increment of apoptosis cells (Annexin V/Propidium Iodide), by the increment of gH2AX foci and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). To understand the relationship between the activation of apoptosis and the effect on cell cycle and to identify hypothetical mechanisms of death, different cell cycle analyses were performed (Propidium Iodide staining). The inhibition of Chk1, deeply changed the cell cycle profile. Indeed in all the cell lines the percentage of cells in S phase and in G2/M phase were reduced by the treatment while the numbers of cells in sub-G1 and G1 phase were increased. The hypothetical function of LY2606368 as a chemosensitizer agent was evaluated combining the compound with different drugs normally used in clinical trials. For each drugs the combination strongly reduced the cell viability when compared to the cytotoxic effect of the single drugs. Moreover the combination showed an additive efficacy in term of induction of DNA damages as showed by the increase number of gH2AX foci and the activation of pChk1 (ser 317). The results found on the cell lines were confirmed also using primary leukemic blast isolated from adult Philadelphia-positive ALL patients. Indeed LY2606368 as single agent or in combination with the Tki, imatinib, was able to deeply reduce the cell viability and to induce DNA damages (gH2AX foci). In conclusion LY2606368 showed a strong cytotoxic activity on B-/T-All cell lines and primary blasts as single agent and in combination with other drugs. In our opinion this data are the basis for a future clinical evaluation of this compound in the treatment of leukemia. Supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:JANSSEN, CELGENE, AMGEN: Consultancy. Martinelli:ROCHE: Consultancy; Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; Ariad: Consultancy; AMGEN: Consultancy; MSD: Consultancy.

scholarly journals Expression of dominant-negative mutant DP-1 blocks cell cycle progression in G1.

1996 ◽  
Vol 16 (7) ◽  
pp. 3698-3706 ◽  
Author(s):  
C L Wu ◽  
M Classon ◽  
N Dyson ◽  
E Harlow
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Dominant Negative ◽  
Functional Domains ◽  
Dominant Negative Mutant ◽  
G1 Arrest ◽  
Wild Type ◽  
Cycle Progression

Unregulated expression of the transcription factor E2F promotes the G1-to-S phase transition in cultured mammalian cells. However, there has been no direct evidence for an E2F requirement in this process. To demonstrate that E2F is obligatory for cell cycle progression, we attempted to inactivate E2F by overexpressing dominant-negative forms of one of its heterodimeric partners, DP-1. We dissected the functional domains of DP-1 and separated the region that facilitate heterodimer DNA binding from the E2F dimerization domain. Various DP-1 mutants were introduced into cells via transfection, and the cell cycle profile of the transfected cells was analyzed by flow cytometry. Expression of wild-type DP-1 or DP-1 mutants that bind to both DNA and E2F drove cells into S phase. In contrast, DP-1 mutants that retained E2F binding but lost DNA binding arrested cells in the G1 phase of the cell cycle. The DP-1 mutants that were unable to bind DNA resulted in transcriptionally inactive E2F complexes, suggesting that the G1 arrest is caused by formation of defective E2F heterodimers. Furthermore, the G1 arrest instigated by these DP-1 mutants could be rescued by coexpression of wild-type E2F or DP protein. These experiments define functional domains of DP and demonstrate a requirement for active E2F complexes in cell cycle progression.

Melatonin inhibits human melanoma cells proliferation and invasion via cell cycle arrest and cytoskeleton remodeling

2020 ◽  
Vol 3 (2) ◽  
pp. 194-209 ◽  
Author(s):  
Ana Carolina Ramos Moreno ◽  
Renata de Freitas Saito ◽  
Manoela Tiago ◽  
Renato Ramos Massaro ◽  
Roberta Liberato Pagni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Cell Swelling ◽  
Cycle Arrest ◽  
Cytoskeleton Remodeling ◽  
Melanoma Cell Lines

Among skin cancers, melanoma has the highest mortality rate. The heterogeneous genetic melanoma background leads to a tumor-propagating capacity particularly important in maintaining therapeutic resistance, and tumor recurrence. The identification of efficient molecules able to control melanoma progress represents an important opportunity for new therapeutic strategies, particularly in combination with the current standard-of-care treatments. In this context, several studies have reported the antitumor effects of melatonin against different types of cancer, including melanoma. Here, we describe the underlying mechanisms associated with melatonin’s activity in human melanoma cell lines, focusing on cell cycle and cytoskeleton remodeling. Interestingly, while melatonin induced melanocyte DNA replication, melanoma cells exhibited cell cycle arrest in the G1-phase. This phenomenon was associated with cyclin-D1 downregulation or p21 overexpression. The efficacy of melatonin on melanoma cells survival and proliferation was detected using the clonogenic assay, with a decrease in both the number and size of colonies. Additionally, melatonin induced a dramatic cytoskeleton remodeling in all melanoma cell lines, leading to a star-like morphology or cell swelling. The role of melatonin on melanoma cytoskeleton was associated with the actin disruption, with thinning and/or broken actin fibers, and weak and/or loss of paxillin along stress fibers. These data support the observed findings that melatonin impairs melanoma invasion in skin reconstructed models. Together, our results suggest that melatonin could be used to control melanoma growth and support basic and clinical studies on melatonin as a promising immunometabolic adjuvant for melanoma therapy.

cJun Is Phosphorylated at Ser73 and Contributes to Cell Cycle Progression in Anaplastic Large Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2620-2620
Author(s):  
Vassiliki Leventaki ◽  
Elias Drakos ◽  
Francois-Xavier Claret ◽  
L. Jeffrey Medeiros ◽  
George Z. Rassidakis
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Cell Lines ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
Large Cell ◽  
S Phase ◽  
Cycle Progression ◽  
Large Cell Lymphoma

Abstract Anaplastic Large Cell Lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). cJun is a member of the activator protein-1 (AP-1) family, which is a group of transcription factors that control cell proliferation, differentiation, growth and apoptosis. The activity of cJun can be regulated by phosphorylation at serine 73 (Ser73) and serine 63 (Ser63) residues of the N-terminal domain. It is believed that cJun promotes cell cycle progression, in part, through downregulation of the cyclin-dependent kinase inhibitor p21. Previous studies have shown high AP-1 activity and cJun overexpression in Hodgkin lymphoma and ALCL (Mathas et al, EMBO J2002; 21:4104). In this study, we assessed for expression of cJun and its Ser73- and Ser63-phosphorylated forms in two ALK+ (Karpas 299 and SU-DHL-1) and one ALK- (Mac2A) ALCL cell lines by western blot analysis, and in 31 ALCL tumors (15 ALK+, 16 ALK-) by immunohistochemistry using tissue microarrays and specific antibodies. To examine the role of cJun in cell survival and proliferation in our in vitro system, ALCL cells were transiently transfected with small interfering RNA (siRNA) specific for cJun. Cell viability, proliferation of viable cells and cell cycle progression from G1 to S-phase were assessed by trypan blue exclusion, MTS and BrdU assays, respectively. All three ALCL cell lines expressed total cJun and Ser73-phosphorylated cJun (Ser73p-cJun) at a high level, whereas Ser63-phosphorylated cJun was expressed at a low level. In addition, all 31 ALCL tumors expressed total cJun in most neoplastic cells. Ser73p-cJun was also detected in all ALCL tumors at a variable level with the percentage of Ser73p-cJun-positive tumor cells ranging from 5% to 95%. By contrast, Ser63p-cJun was detected rarely in tumor cells. Transient transfection of ALCL cells with specific siRNA resulted in almost complete silencing of total cJun expression and absence of Ser73p-cJun expression, which was associated with decreased cell viability and a substantial (40%) decrease of cell growth. cJun silencing also resulted in cell cycle arrest as shown by decreased S-phase fraction. These cell cycle changes were associated with a marked increase of p21 levels and downregulation of cyclin D2 and D3. In conclusion, cJun is highly phosphorylated at serine 73 in ALCL cell lines and tumors and may contribute to cell cycle progression. Targeting cJun expression or phosphorylation using gene therapy approaches may represent a novel therapeutic strategy for patients with ALCL.

Bim Is Required to Sensitize Mantle Cell Lymphoma Cells for Killing by Bortezomib, but Not Ara-C, by Selective Inhibition of CDK4/CDK6

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1655-1655
Author(s):  
Xiangao Huang ◽  
Maurizio Di Liberto ◽  
Jamieson Bretz ◽  
David Chiron ◽  
Peter Martin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Research Funding ◽  
Cell Cycle Progression ◽  
Phase Synchronization ◽  
Selective Inhibition ◽  
S Phase ◽  
G1 Arrest ◽  
Cycle Progression ◽  
Mantle Cell

Abstract Abstract 1655 Mantle cell lymphoma (MCL) is characterized by aberrant cyclin D1 expression due to the t (11: 14) translocation. In conjunction with elevation of CDK4/CDK6, this promotes cell cycle progression through G1 and unrestrained cell proliferation. As MCL remains incurable despite initial response to therapy, mechanism- and genome-based therapies that both control the cell cycle and enhance cytotoxic killing are urgently needed. We have recently developed such a regimen by inhibition of CDK4/CDK6 with PD 0332991 (PD), a selective inhibitor of CDK4 and CDK6 that is also potent, reversible and orally bioavailable. We demonstrate that 1) inhibition of CDK4/CDK6 with PD leads to early G1 arrest; 2) upon release of the G1 block, synchronous cell cycle progression to S phase occurs; and 3) S phase synchronization following prolonged early G1 arrest (pG1-S) sensitizes MCL cells to killing by diverse clinically relevant agents at reduced doses, including proteasome inhibitors bortezomib and carfilzomib, and the nucleoside analog Ara-C (cytarabine), both in vitro and in a mouse model of MCL. These findings implicate a unified mechanism for cell cycle sensitization of cytotoxic killing. To elucidate the underpinning mechanism, we show that sensitization to cytotoxic killing by CDK4/CDK6 inhibition requires an intact Rb, the substrate of CDK4/CDK6, but is independent of p53. Gene expression profiling and quantitative RNA and protein analyses further demonstrate that prolonged inhibition of CDK4/CDK6 with PD halts the gene expression program in early G1 and depletes the expression of genes programmed for other phases of the cell cycle, such as cyclin A (G1/S), thymidine kinase (S), CDK1 and cyclin B (G2/M) and selective metabolic genes. Removal of PD restores the CDK4/CDK6 activities and the expression of scheduled cell cycle genes but leaves many others in the pG1 state. This leads to S phase synchronization with impaired metabolism. Accordingly, the magnitude of bortezomib and Ara-C killing in pG1-S greatly exceeds the enrichment of S phase cells. Selective inhibition of CDK4/CDK6, therefore, sensitizes MCL cells for cytotoxic killing in S phase synchronization through induction of a persistent metabolic imbalance in prior pG1. pG1 alone induces caspase activation moderately in MCL cells, but markedly augments apoptosis induced by either bortezomib or Ara-C in pG1-S. This enhancement of apoptosis is apparently mediated by an alteration of the ratios of pro-apoptotic BH3-only proteins (Bim, Noxa and Puma) to anti-apoptotic proteins (Mcl-1, Bcl-2 and Bcl-xL), which lowers the threshold for caspase-9 activation. Importantly, Bim is selectively required to sensitize MCL cells for killing by bortezomib, but not Ara-C, at low doses as indicated in studies of Bim-deficient MCL cell lines. Corroborating these findings, loss of one allele of Bim attenuates the enhancement of bortezomib killing in pG1-S in untransformed primary mouse B cells after activation by BCR and CD40 signaling. Thus, the synergistic actions of PD-bortezomib and PD-AraC in MCL therapy are distinguishable by the requirement for Bim. Furthermore, we found that the three Bim isoforms are expressed at variable levels but undetected in 30% of primary MCL tumor cells, consistent with the reported mutations and bi-allelic deletion of Bim (BCL2L11) in MCL. RNA-Seq analysis of samples from patients enrolled in a phase I study of PD in combination with bortezomib in MCL further reveals that the mutation burden in BCL2L11 is ∼3-fold higher in a clinically non-responder compared with a responder. Collectively, our data demonstrate that by halting scheduled gene expression in prolonged early G1 arrest, selective and reversible inhibition of CDK4/CDK6 provides a mechanism-based strategy to sensitize MCL cells for cytotoxic killing by bortezomib, Ara-C, and potentially other emerging agents. By lowering the threshold for caspase activation, Bim is selectively required for sensitization to killing by low dose bortezomib, but not Ara-C, and may serve as a biomarker for genome-based selection of cytotoxic partners in therapeutic targeting of CDK4/CDK6 in MCL. Disclosures: Martin: Millennium Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Smith:Pfizer: Research Funding; Millenium: Research Funding. Leonard:Pfizer, Inc.: Consultancy; Millenium: Consultancy; Johnson and Johnson: Consultancy; Onyx: Consultancy. Chen-Kiang:Pfizer, Inc.: Research Funding.

Sign in / Sign up

Export Citation Format

Share Document