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. 2016 Aug 30;7(35):56664-56675.
doi: 10.18632/onco_target.10994.

Met promotes the formation of double minute chromosomes induced by Sei-1 in NIH-3T3 murine fibroblasts

Yantao Bao  1 , Jia Liu  1 , Jia You  1 , Di Wu  1 , Yang Yu  1   2 , Chang Liu  1 , Lei Wang  1   3 , Fei Wang  1 , Lu Xu  1 , Liqun Wang  1 , Nan Wang  1 , Xing Tian  1 , Falin Wang  1 , Hongbin Liang  1 , Yating Gao  1 , Xiaobo Cui  1 , Guohua Ji  1 , Jing Bai  1 , Jingcui Yu  4 , Xiangning Meng  1 , Yan Jin  1 , Wenjing Sun  1 , Xin-Yuan Guan  5   6 , Chunyu Zhang  1 , Songbin Fu  1   7
Affiliations

Met promotes the formation of double minute chromosomes induced by Sei-1 in NIH-3T3 murine fibroblasts

Yantao Bao et al. Onco_target. .

Abstract

Background: Sei-1 is an oncogene capable of inducing double minute chromosomes (DMs) formation. DMs are hallmarks of amplification and contribute to oncogenesis. However, the mechanism of Sei-1 inducing DMs formation remains unelucidated.

Results: DMs formation significantly increased during serial passage in vivo and gradually decreased following culture in vitro. micro nuclei (MN) was found to be responsible for the reduction. Of the DMs-carrying genes, Met was found to be markedly amplified, overexpressed and highly correlated with DMs formation. Inhibition of Met signaling decreased the number of DMs and reduced the amplification of the DMs-carrying genes. We identified a 3.57Mb DMs representing the majority population, which consists of the 1.21 Mb AMP1 from locus 6qA2 and the 2.36 Mb AMP2 from locus 6qA2-3.

Materials and methods: We employed NIH-3T3 cell line with Sei-1 overexpression to monitor and characterize DMs in vivo and in vitro. Array comparative genome hybridization (aCGH) and fluorescence in situ hybridization (FISH) were performed to reveal amplification regions and DMs-carrying genes. Metaphase spread was prepared to count the DMs. Western blot and Met inhibition rescue experiments were performed to examine for involvement of altered Met signaling in Sei-1 induced DMs. Genomic walking and PCR were adopted to reveal DMs structure.

Conclusions: Met is an important promotor of DMs formation.

Keywords: DMs; Sei-1; amplification; in vivo passage; met.

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Conflict of interest statement

The authors declare No potential conflicts of interest.

Figures

Figure 1
Figure 1. DMs population evolution in vivo and in vitro
(A) Western blot results show Sei-1 overexpression in NIH-3T3 cell clone Sei-1/NIH-3T3 compared with Vec. Upper, quantification of western blot results (n = 3), lower, representative western blot image. (B) Flow chart of in vivo and in vitro passage procedure. Three parallel procedures were employed, each mouse bore two tumor implants. (C) Metaphase spread images of primary cells CPX1, CPX3 and CPX6. Red arrows indicated the DMs. (E) Metaphase spread of CPX6 after four, eight, and 12 weeks of in vitro passage. (D) DMs count in CPX1, CPX3 and CPX6, for each group, n = 110 from three independent studies. (F) DMs number per karyotype in primary cells in four, eight, and 12 weeks of in vitro passage, n = 100 for each group. (G) FISH assays that show DMs signals in MNs after four, eight, and 12 weeks of in vitro passage. (H) The percentage of MNs with different staining states—“–”, no DMs signal, “+”, 0–50% DMs signal, and “++”–50%–100% DMs signals after four, eight, and 12 weeks of in vitro passage. Scale Bar, 10 μm. ***P < 0.001.
Figure 2
Figure 2. Identification of amplicons and amplified genes on DMs
(A) Schematic of aCGH (Sei-1/NIH-3T3 vs CPX6) shows amplicons on chr6qA2-3; amplified BAC probes were represented as red dots, deleted probes were represented as green dots, and the normal probes as black dots. The BAC clones that were employed to validate the amplicons were labeled in corresponding positions of chromosomes. (B) Representative FISH images using the BAC clones that are shown in red; scale bar, 10 μm, quantitative real-time PCR results of DNA amplification level (C) and mRNA expression level (D) show amplification and overexpression of genes carried on DMs.
Figure 3
Figure 3. Met signaling was activated during in vivo passage
(A) Western blot results show increases of Met/PI3K/Akt pathway from Sei-1/NIH-3T3, CPX3 to CPX6 during in vivo passage. (B) Quantification of protein level in A, n = 3. (C) Met phosphorylation was increased in the presence (“+”) of Hgf treatment from Sei-1/NIH-3T3, CPX3 to CPX6, at a dosage of 20 ng/ml, and absence (“–“) of Hgf treatment from Sei-1/NIH-3T3, CPX3 to CPX6 by in vivo passage. (D) Quantification of protein level in C with (+) or without (−) Hgf treatment, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 4
Figure 4. Met inhibition caused a loss in DMs
Western blot results show effective inhibition in CPX3 (A) and CPX6 (B) for 24 and 48 hours with 4 × 10−4 μg/ml Met inhibitor treatment. Quantification data are shown in (C) (CPX3) and (D) (CPX6), n = 3. Scatter plot show a significant decrease in the DMs population 12, 24, and 36 hours with Met inhibition in (E) CPX3 and (F) CPX6, for each group, n = 100 from three independent studies. Quantitative real-time PCR results show decreased expression levels of DMs-carrying genes with Met inhibition in (G) CPX3 and (H) CPX6. A two-tailed student's t-test was employed as statistics; *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5. DMs structures consist of two individual amplicons
(A) The boundaries of AMP1 and AMP2 were defined by quantitative real-time PCR for BP1, 2 and 4, and semi-quantitative PCR for BP3. (B) Blunt-end joints were identified via Sanger sequencing. AMP1 is shown in red and AMP2 is shown in green. (C) A schematic of DMs structures with DMs-carrying genes marked in the corresponding positions; the arrows indicate the transcription direction of each gene.
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