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. 2011 Mar 16;10:28.
doi: 10.1186/1476-4598-10-28.

Overexpression of Aurora-A in Primary Cells Interferes With S-phase Entry by Diminishing Cyclin D1 Dependent Activities

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Free PMC article

Overexpression of Aurora-A in Primary Cells Interferes With S-phase Entry by Diminishing Cyclin D1 Dependent Activities

Florian Jantscher et al. Mol Cancer. .
Free PMC article

Abstract

Background: Aurora-A is a bona-fide oncogene whose expression is associated with genomic instability and malignant transformation. In several types of cancer, gene amplification and/or increased protein levels of Aurora-A are a common feature.

Results: In this report, we describe that inhibition of cell proliferation is the main effect observed after transient overexpression of Aurora-A in primary human cells. In addition to the known cell cycle block at the G2/M transition, Aurora-A overexpressing cells fail to overcome the restriction point at the G1/S transition due to diminished RB phosphorylation caused by reduced Cyclin D1 expression. Consequently, overexpression of Cyclin D1 protein is able to override the Aurora-A mediated G1 block. The Aurora-A mediated cell cycle arrest in G2 is not influenced by Cyclin D1 and as a consequence cells accumulate in G2. Upon deactivation of p53 part of the cells evade this premitotic arrest to become aneuploid.

Conclusion: Our studies describe that an increase of Aurora-A expression levels on its own has a tumor suppressing function, but in combination with the appropriate altered intracellular setting it might exert its oncogenic potential. The presented data indicate that deactivation of the tumor suppressor RB is one of the requirements for overriding a cell cycle checkpoint triggered by increased Aurora-A levels.

Figures

Figure 1
Figure 1
Influence of Aurora-A overexpression on cell cycle. (A) Wi-38 cells were infected with adenoviruses expressing either Aurora-A or control (lacZ) proteins. Overexpressed protein levels were compared to T98G (glioblastoma), VM24, and VM7 (melanoma) cell lines. (B) Wi-38 cells overexpressing Aurora-A were tested in a growth curve experiment against cells infected with a control virus. The results of two independent experiments are depicted. (C) Logarithmically growing Wi-38 cells were infected with the viruses expressing the indicated proteins and DNA content was measured 24, 48 and 72 hours after infection. The time curve shows the percentage of cells in G1 and S-phase from a representative experiment. The histograms and the respective cell cycle analysis of the 72 hours time point is depicted. Bars illustrate means and standard deviation of four independent experiments. Statistical analysis was done using an unpaired, two-sided t-test. * P < 0.05.
Figure 2
Figure 2
G1 cell cycle arrest mediated by Aurora-A wildtype and K162R. (A) Serum starved Wi-38 cells were infected with adenoviruses expressing either Aurora-A or control (lacZ) proteins. Cell were induced by serum addition and protein levels were compared to those of equally treated T98G cells 26 hours post induction (B) Arrested Wi-38 cells were infected with adenoviruses expressing the proteins indicated. 48 hours after infection serum was added and PI-stainings were collected. A representative of three experiments is shown. (C) Cells were treated as in (B) and a 3H-thymidine incorporation assay was performed. Bars depict means and SD of three independent experiments.
Figure 3
Figure 3
Importance of known Aurora-A interactions for cell cycle inhibition. (A) Wi-38 cells were arrested in G0 by serum deprivation and infected with adenoviruses expressing Aurora-A or control virus expressing lacZ and analyzed by immunoblotting with the antibodies indicated. (B) Quiescent cells were infected with adenoviruses expressing the indicated proteins and analyzed by immunoblotting and PI staining at time points 0 and 26 hours after serum addition. (C) Serum arrested cells expressing Aurora-A or lacZ were transfected with the indicated siRNA 24 hours prior to serum addition. At the indicated time points cells were harvested and analyzed as in (B). (D) Serum deprived cells infected with control or Aurora-A expressing viruses were harvested at the indicated times and analyzed by immunoblotting with antibodies recognizing phosphorylation of ERK1/2 and ribosomal protein S6, respectively. For all illustrations, representatives of at least two independent experiments are shown.
Figure 4
Figure 4
Identification of modulated gene expression caused by Aurora-A expression during G0/G1 progression. 24 hours after serum removal, arrested Wi-38 cells were infected with adenoviruses expressing either Aurora-A or lacZ (control). Serum was added 48 hours after infection and cells were harvested at the times indicated. Total RNA was extracted and Clonetech human Atlas 1.2 cDNA expression array analyses were performed. (A) A list of the genes, which fail to respond to serum addition in Aurora-A overexpressing cells is presented in the upper panel. Ratio given indicates the difference in mRNA expression compared to controls. The lower panel highlights the region of the array containing the probes for Cyclin D1 and p19INK4d. (B) Verification of the array data was done using Northern blot analysis with a Cyclin D1 probe spanning the first 350 nucleotides of the Cyclin D1 coding sequence and a probe for GAPDH from nucleotides 192 to 549 for normalization. One of 3 Northern blots from independent experiments is shown. The expression level ratios of Cyclin D1 were calculated after densitometric analysis using ImageQuant software (Molecular Dynamics, Sunnyvale, CA) and normalized to GAPDH. Serum-deprived control cells were set to 1. (C) Immunoblot of equally treated cells was performed and assayed for Cyclin D1 protein expression. (D) At the indicated times, immunoblotting of serum released cells expressing either lacZ (control) or Aurora-A was performed with the antibodies indicated.
Figure 5
Figure 5
Influence of Cyclin D1 co-expression along with Aurora-A on G1/S arrest. G0 arrested Wi-38 cells were infected with the viruses expressing the indicated proteins 24 hours after serum depletion. 48 hours after infection, serum was added. (A) At the indicated time points, PI-stainings were performed. (B) In parallel, cell extracts were analyzed by immunoblotting with the indicated antibodies. A representative experiment of at least three experiments is shown. (C) Serum arrested cells expressing Aurora-A or lacZ were transfected with the control or Rb specific siRNA. 26 hours after serum release, cells were analyzed by immunoblotting and PI-stainings.
Figure 6
Figure 6
Long term effects of Aurora-A and Cyclin D1 co-expression. (A) Logarithmically growing Wi-38 cells were infected with the adenoviruses indicated. Three days after infection, cells were analyzed by PI-staining. One of two data sets is presented. (B) A growth curve experiment was performed with logarithmically growing Wi-38 infected with the viruses indicated. Data of two independent experiments are shown. (C) Serum starved Wi-38 cells were infected with adenoviruses expressing control (lacZ), Aurora-A, Cyclin D1 or a combination of these proteins. Cells were induced by serum addition and analyzed by PI-staining after 48 hours. A representative of three experiments is depicted. (D) Wi-38 cells were treated as in (C), but infected with the indicated combinations of Aurora-A, Cyclin D1 and p53V143A expressing adenoviruses. After 48 and 72 hours, cells were analyzed by PI-staining. Bars depict the percentage of cells with a DNA content >4N. A representative of two experiments is shown.

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