SU9516, a cyclin-dependent kinase 2 inhibitor, promotes accumulation of high molecular weight E2F complexes in human colon carcinoma cells

Bo Yu, Maureen E. Lane, Scott Wadler

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The E2F family plays a critical role in the expression of genes required for entry into and progression through S phase. E2F-mediated transcription is repressed by the tumor suppressor retinoblastoma protein (pRb), which results in sequestration of E2F in a multiprotein complex that includes pRb. Derepression of E2F results from a series of complex phosphorylation events mediated by cyclin D/cdk4 and cyclin E/cdk2. We have employed a novel 3-substituted indolinone compound, 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), which selectively inhibits cdk2 activity (Lane et al., Cancer Res 2001;61:6170-7) to investigate these events. Electrophoretic mobility gel shift assays were performed on SU9516-treated and -untreated HT-29, SW480, and RKO human colon cancer cell extracts. Treatment with 5μM SU9516 prevented dissociation of pRb from E2F1 in all cell lines (HT-29>RKO>SW480). Treatment effects were time-dependent, demonstrating greater inhibition at 48hr versus 24hr in HT-29 cells. Furthermore, E2F species were sequestered in complexes with p107, p130, DP-1, and cyclins A and E. After a 24-hr treatment with 5μM SU9516, cyclin D1 and cdk2 levels decreased by 10-60%. These findings delineate a previously undescribed mechanism for SU9516-mediated cell growth arrest through down-regulation of cyclin D1, inhibition of cdk2 levels and activity, and pan-sequestration of E2F.

Original languageEnglish (US)
Pages (from-to)1091-1100
Number of pages10
JournalBiochemical Pharmacology
Volume64
Issue number7
DOIs
StatePublished - Oct 1 2002

Fingerprint

Cyclin-Dependent Kinase 2
Colon
Molecular Weight
Molecular weight
Cells
Carcinoma
Cyclin E
Cyclin D1
Cyclin D
Tumor Suppressor Proteins
Cyclin A
Multiprotein Complexes
Retinoblastoma Protein
HT29 Cells
Electrophoretic mobility
Phosphorylation
Cell growth
Electrophoretic Mobility Shift Assay
Transcription
Cell Extracts

Keywords

  • Cyclin
  • Cyclin-dependent kinase
  • Cyclin-dependent kinase inhibitor
  • E2F
  • Retinoblastoma protein

ASJC Scopus subject areas

  • Pharmacology

Cite this

SU9516, a cyclin-dependent kinase 2 inhibitor, promotes accumulation of high molecular weight E2F complexes in human colon carcinoma cells. / Yu, Bo; Lane, Maureen E.; Wadler, Scott.

In: Biochemical Pharmacology, Vol. 64, No. 7, 01.10.2002, p. 1091-1100.

Research output: Contribution to journalArticle

@article{f29874bf79084ed9b338a0ddd1aa27fb,
title = "SU9516, a cyclin-dependent kinase 2 inhibitor, promotes accumulation of high molecular weight E2F complexes in human colon carcinoma cells",
abstract = "The E2F family plays a critical role in the expression of genes required for entry into and progression through S phase. E2F-mediated transcription is repressed by the tumor suppressor retinoblastoma protein (pRb), which results in sequestration of E2F in a multiprotein complex that includes pRb. Derepression of E2F results from a series of complex phosphorylation events mediated by cyclin D/cdk4 and cyclin E/cdk2. We have employed a novel 3-substituted indolinone compound, 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), which selectively inhibits cdk2 activity (Lane et al., Cancer Res 2001;61:6170-7) to investigate these events. Electrophoretic mobility gel shift assays were performed on SU9516-treated and -untreated HT-29, SW480, and RKO human colon cancer cell extracts. Treatment with 5μM SU9516 prevented dissociation of pRb from E2F1 in all cell lines (HT-29>RKO>SW480). Treatment effects were time-dependent, demonstrating greater inhibition at 48hr versus 24hr in HT-29 cells. Furthermore, E2F species were sequestered in complexes with p107, p130, DP-1, and cyclins A and E. After a 24-hr treatment with 5μM SU9516, cyclin D1 and cdk2 levels decreased by 10-60{\%}. These findings delineate a previously undescribed mechanism for SU9516-mediated cell growth arrest through down-regulation of cyclin D1, inhibition of cdk2 levels and activity, and pan-sequestration of E2F.",
keywords = "Cyclin, Cyclin-dependent kinase, Cyclin-dependent kinase inhibitor, E2F, Retinoblastoma protein",
author = "Bo Yu and Lane, {Maureen E.} and Scott Wadler",
year = "2002",
month = "10",
day = "1",
doi = "10.1016/S0006-2952(02)01264-9",
language = "English (US)",
volume = "64",
pages = "1091--1100",
journal = "Biochemical Pharmacology",
issn = "0006-2952",
publisher = "Elsevier Inc.",
number = "7",

}

TY - JOUR

T1 - SU9516, a cyclin-dependent kinase 2 inhibitor, promotes accumulation of high molecular weight E2F complexes in human colon carcinoma cells

AU - Yu, Bo

AU - Lane, Maureen E.

AU - Wadler, Scott

PY - 2002/10/1

Y1 - 2002/10/1

N2 - The E2F family plays a critical role in the expression of genes required for entry into and progression through S phase. E2F-mediated transcription is repressed by the tumor suppressor retinoblastoma protein (pRb), which results in sequestration of E2F in a multiprotein complex that includes pRb. Derepression of E2F results from a series of complex phosphorylation events mediated by cyclin D/cdk4 and cyclin E/cdk2. We have employed a novel 3-substituted indolinone compound, 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), which selectively inhibits cdk2 activity (Lane et al., Cancer Res 2001;61:6170-7) to investigate these events. Electrophoretic mobility gel shift assays were performed on SU9516-treated and -untreated HT-29, SW480, and RKO human colon cancer cell extracts. Treatment with 5μM SU9516 prevented dissociation of pRb from E2F1 in all cell lines (HT-29>RKO>SW480). Treatment effects were time-dependent, demonstrating greater inhibition at 48hr versus 24hr in HT-29 cells. Furthermore, E2F species were sequestered in complexes with p107, p130, DP-1, and cyclins A and E. After a 24-hr treatment with 5μM SU9516, cyclin D1 and cdk2 levels decreased by 10-60%. These findings delineate a previously undescribed mechanism for SU9516-mediated cell growth arrest through down-regulation of cyclin D1, inhibition of cdk2 levels and activity, and pan-sequestration of E2F.

AB - The E2F family plays a critical role in the expression of genes required for entry into and progression through S phase. E2F-mediated transcription is repressed by the tumor suppressor retinoblastoma protein (pRb), which results in sequestration of E2F in a multiprotein complex that includes pRb. Derepression of E2F results from a series of complex phosphorylation events mediated by cyclin D/cdk4 and cyclin E/cdk2. We have employed a novel 3-substituted indolinone compound, 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), which selectively inhibits cdk2 activity (Lane et al., Cancer Res 2001;61:6170-7) to investigate these events. Electrophoretic mobility gel shift assays were performed on SU9516-treated and -untreated HT-29, SW480, and RKO human colon cancer cell extracts. Treatment with 5μM SU9516 prevented dissociation of pRb from E2F1 in all cell lines (HT-29>RKO>SW480). Treatment effects were time-dependent, demonstrating greater inhibition at 48hr versus 24hr in HT-29 cells. Furthermore, E2F species were sequestered in complexes with p107, p130, DP-1, and cyclins A and E. After a 24-hr treatment with 5μM SU9516, cyclin D1 and cdk2 levels decreased by 10-60%. These findings delineate a previously undescribed mechanism for SU9516-mediated cell growth arrest through down-regulation of cyclin D1, inhibition of cdk2 levels and activity, and pan-sequestration of E2F.

KW - Cyclin

KW - Cyclin-dependent kinase

KW - Cyclin-dependent kinase inhibitor

KW - E2F

KW - Retinoblastoma protein

UR - http://www.scopus.com/inward/record.url?scp=0036776132&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036776132&partnerID=8YFLogxK

U2 - 10.1016/S0006-2952(02)01264-9

DO - 10.1016/S0006-2952(02)01264-9

M3 - Article

C2 - 12234612

AN - SCOPUS:0036776132

VL - 64

SP - 1091

EP - 1100

JO - Biochemical Pharmacology

JF - Biochemical Pharmacology

SN - 0006-2952

IS - 7

ER -