Detection and Analysis of Living, Growth-Inhibited Mammalian Cells Following Transfection

Zeng, Yi‐Xin; Somasundaram, Kumaravel; Prabhu, Nita S.; Krishnadasan, Ravitharan; El‐Deiry, Wafik S.

doi:10.2144/97231bm19

Cited by 9 publications

(10 citation statements)

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“…For these experiments, cotransfection of the Green Fluorescent Protein was used as a marker to identify transfected cells ± GFP appears not to interfere with cell growth and function ± (Zeng et al, 1997).…”

Section: Resultsmentioning

confidence: 99%

A role for protein kinase CK2 in cell proliferation: evidence using a kinase-inactive mutant of CK2 catalytic subunit α

Lebrin¹,

Chambaz²,

Bianchini³

2001

Oncogene

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Protein kinase CK2 is an ubiquitous and pleiotropic Ser/ Thr protein kinase composed of two catalytic (a and/or a') and two regulatory (b) subunits generally combined to form a 2 b 2 , aa'b 2 , or a' 2 b 2 heterotetramers. To gain more insight into the role of CK2 in the control of proliferation in mammalian cells, overexpression of isolated CK2 subunits a, a', or b was carried out in two ®broblast cell lines: NIH3T3 and CCL39. To interfere with CK2 cellular functions, cells were also transfected with a kinase-inactive mutant of CK2a catalytic subunit: CK2a-K68A. In NIH3T3 cells, overexpression of either wild-type subunit (a, a' or b) had no eect on cell proliferation. In contrast, overexpression of the CK2a kinase-de®cient mutant induced a marked inhibition of cell proliferation. This resulted from a defect in G1/S progression as demonstrated in transient transfection experiments in both NIH3T3 and CCL39 cells using BrdU incorporation measurements and in CCL39 clones stably overexpressing the CK2a-K68A mutant by growth curve analysis. We demonstrated that the kinase-negative mutant has the capacity to integrate the endogenous CK2 subunit pool both as an isolated kinase-inactive a subunit and as associated to the b subunit in a kinase-inactive tetramer. Finally we showed that expression of the kinase-inactive mutant interferes with phosphorylation of an endogenous CK2 substrate; we speculate that optimal phosphorylation of target proteins by CK2 is required to achieve optimal cell cycle progression.

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Section: Resultsmentioning

confidence: 99%

A role for protein kinase CK2 in cell proliferation: evidence using a kinase-inactive mutant of CK2 catalytic subunit α

Lebrin¹,

Chambaz²,

Bianchini³

2001

Oncogene

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“…Transfections were carried out as previously described (Zeng et al, 1997). In brief before transfection, cells were seeded at 1610 6 cells/6 cm dish.…”

Section: Transfections and Reporter Assaysmentioning

confidence: 99%

“…Serum containing media was added at 4 h after transfection. Cells were harvestted 24 h after transfection and assayed for luciferase and b-galactosidase activity as previously described (Oliner et al, 1993;Zeng et al, 1997).…”

Section: Transfections and Reporter Assaysmentioning

confidence: 99%

Wild-type p53 transactivates the KILLER/DR5 gene through an intronic sequence-specific DNA-binding site

2000

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KILLER/DR5, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor gene, has been shown to be induced by DNA damaging agents and radiation in a p53-dependent manner. Although TRAIL is a potential therapeutic agent for cancer, the induction mechanism of its receptors is poorly understood. Here we show the identi®cation of three p53 DNA-binding sites in the KILLER/DR5 genomic locus located upstream (BS1; 70.82 Kb) of the ATG site, within Intron 1 (BS2; +0.25 Kb downstream of the ATG) and within Intron 2 (BS3; +1.25 Kb downstream of the ATG). A modi®ed p53-binding and immunoselection protocol using a wildtype p53-expressing adenovirus vector (Ad-p53) was used to identify the binding sites and to show that each binding site can bind speci®cally to wild-type p53 protein (wt-p53). A reporter assay revealed that only BS2 could enhance luciferase expression driven by a basal promoter. We constructed a reporter plasmid carrying the genomic regulatory region of KILLER/DR5 including the three p53 DNA-binding sites but no additional basal promoter. The genomic fragment showed basal transcriptional activity which was induced by wt-p53 but not by mutant p53, and human papilloma virus E6 inhibited the p53-dependent activation. Mutation of BS2 abrogated not only the binding activity of wt-p53 but also the induction of the KILLER/DR5 genomic promoter-reporter gene, indicating that BS2 is responsible for the p53-dependent transactivation of KILLER/ DR5. In p53-wild-type but not -mutant or -null cell lines, doxorubicin treatment stabilized p53 protein, and increased speci®c binding to BS2 as revealed by EMSA, and upregulated the KILLER/DR5 promoter-luciferase reporter gene. These results suggest that the transactivation of KILLER/DR5 is directly regulated by exogenous or endogenous wt-p53 and establishes KILLER/DR5 as a p53 target gene that can signal apoptotic death.

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“…To address this issue, studies were done in which LP-ROSE-179 cells were cotransfected with a GFP expression vector and either control or SF-1 expression vector. In this experimental design, cells that expressed GFP were considered to be cotransfected with either control or SF-1 expression vector (24,28). These cotransfection studies showed that the number of cells transfected with control vector increased nearly 2-fold when cultured with serum (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To confirm the studies on populations of transfected cells, LP-ROSE-179 cells were plated in 8-chamber lab-tek slides and then cotransfected with a green fluorescent protein (GFP) expression vector (24). In this study, the GFP expression vector (pEGFP-C1, CLONTECH, Palo Alto, CA) was diluted to a final concentration of 1.25 ng/l and then was transfected with either control or SF-1 expression vector, as previously described.…”

Section: Assessment Of Mitosis or Apoptosis Of Individual Transfectedmentioning

confidence: 99%

Steroidogenic Factor-1 Regulates the Rate of Proliferation of Normal and Neoplastic Rat Ovarian Surface Epithelial Cellsin Vitro¹

et al. 1998

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Steroidogenic factor-1 (SF-1) is a transcription factor that is expressed by many cell types within the ovary and has been shown to inhibit granulosa cell proliferation. The present studies were designed to determine whether: 1) SF-1 is expressed by primary and transformed rat ovarian surface epithelial cells (i.e. ROSE cells); and 2) SF-1 expression effects the proliferation of both normal and neoplastic ROSE cells. These studies used immature, gonadotropinprimed and mature rat ovaries, as well as ROSE-179 cells from early passages (EP) and late passages (LP), T-sv-40 transformed ROSE cells, and T-ras transformed ROSE cells. In situ hybridization studies demonstrated that SF-1 was detected in the surface epithelium of rat ovaries, independent of age or gonadotropin treatment. Further, Northern blot and quantitative in situ hybridization studies revealed that significant amounts of SF-1 messenger RNA (mRNA) were present in EP-ROSE-179 cells but not in the other cell lines. Interestingly, EP-ROSE-179 cells proliferated at a significantly slower rate than the other cell lines. Further, SF-1 mRNA levels were higher in EP-ROSE-179 cells in the G 0 /G 1 stage than in the S-, G 2 /M stage of the cell cycle. These observations suggest that a cause and effect relationship exists between the level of SF-1 expression and cell proliferation. To test this hypothesis, LP, T-sv-40, and T-ras ROSE cells were transfected with either control vector or SF-1 expression vector.Forty-eight hours after transfection, SF-1 expression was assessed by in situ hybridization, and the fold increase in cell number/24 h was determined. For each cell line, about 30% of the cells were successfully transfected. The fold increase in the number of cells observed after transfection with the SF-1 expression vector was significantly less than the increase in cell number after transfection with the control vector (P Ͻ 0.05). To confirm that the forced expression of SF-1 prevented proliferation, LP cells were cotransfected with a green fluorescent protein (GFP) expression vector and either control vector or SF-1 expression vector. This study demonstrated that virtually none of the GFP/SF-1-transfected cells proliferated over a 24-h period, whereas GFP/Control vector-transfected cells proliferated. Further, approximately 40% of the GFP/SF-1-transfected cells underwent apoptosis after 24 h of culture in serum-supplemented medium. These data demonstrate that: 1) normal ovarian surface epithelial cells express SF-1; 2) SF-1 is also expressed by EP-ROSE-179 cells, but its expression seems to be suppressed when the cells enter the cell cycle; 3) LP-, T-sv, and T-ras ROSE cells do not express SF-1 mRNA; and 4) the inability to express SF-1 is associated with an increase in cell proliferation. Finally, forced SF-1 expression interferes with seruminduced proliferation and leads to apoptosis. (Endocrinology 139: [4663][4664][4665][4666][4667][4668][4669][4670][4671] 1998)

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Detection and Analysis of Living, Growth-Inhibited Mammalian Cells Following Transfection

Cited by 9 publications

References 0 publications

A role for protein kinase CK2 in cell proliferation: evidence using a kinase-inactive mutant of CK2 catalytic subunit α

A role for protein kinase CK2 in cell proliferation: evidence using a kinase-inactive mutant of CK2 catalytic subunit α

Wild-type p53 transactivates the KILLER/DR5 gene through an intronic sequence-specific DNA-binding site

Steroidogenic Factor-1 Regulates the Rate of Proliferation of Normal and Neoplastic Rat Ovarian Surface Epithelial Cellsin Vitro¹

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Detection and Analysis of Living, Growth-Inhibited Mammalian Cells Following Transfection

Cited by 9 publications

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A role for protein kinase CK2 in cell proliferation: evidence using a kinase-inactive mutant of CK2 catalytic subunit α

A role for protein kinase CK2 in cell proliferation: evidence using a kinase-inactive mutant of CK2 catalytic subunit α

Wild-type p53 transactivates the KILLER/DR5 gene through an intronic sequence-specific DNA-binding site

Steroidogenic Factor-1 Regulates the Rate of Proliferation of Normal and Neoplastic Rat Ovarian Surface Epithelial Cellsin Vitro1

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Steroidogenic Factor-1 Regulates the Rate of Proliferation of Normal and Neoplastic Rat Ovarian Surface Epithelial Cellsin Vitro¹