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HIPK2 deficiency causes chromosomal instability by cytokinesis failure and increases tumorigenicity.

Valente D, Bossi G, Moncada A, Tornincasa M, Indelicato S, Piscuoglio S, Karamitopoulou ED, Bartolazzi A, Pierantoni GM, Fusco A, Soddu S, Rinaldo C - Oncotarget (2015)

Bottom Line: In particular, HIPK2 is required to complete cytokinesis and impaired HIPK2 expression results in cytokinesis failure and tetraploidization.Importantly, we found a significant correlation among reduced HIPK2 expression, high grade of malignancy, and high nuclear size, a marker of increased ploidy.Overall, these results indicate that HIPK2 acts as a caretaker gene, whose inactivation increases tumorigenicity and causes CIN by cytokinesis failure.

View Article: PubMed Central - PubMed

Affiliation: Experimental Oncology Laboratory, Regina Elena National Cancer Institute, Rome, Italy.

ABSTRACT
HIPK2, a cell fate decision kinase inactivated in several human cancers, is thought to exert its oncosuppressing activity through its p53-dependent and -independent apoptotic function. However, a HIPK2 role in cell proliferation has also been described. In particular, HIPK2 is required to complete cytokinesis and impaired HIPK2 expression results in cytokinesis failure and tetraploidization. Since tetraploidy may yield to aneuploidy and chromosomal instability (CIN), we asked whether unscheduled tetraploidy caused by loss of HIPK2 might contribute to tumorigenicity. Here, we show that, compared to Hipk2+/+ mouse embryo fibroblasts (MEFs), hipk2- MEFs accumulate subtetraploid karyotypes and develop CIN. Accumulation of these defects inhibits proliferation and spontaneous immortalization of primary MEFs whereas increases tumorigenicity when MEFs are transformed by E1A and Harvey-Ras oncogenes. Upon mouse injection, E1A/Ras-transformed hipk2- MEFs generate tumors with genetic alterations resembling those of human cancers derived by initial tetraploidization events, such as pancreatic adenocarcinoma. Thus, we evaluated HIPK2 expression in different stages of pancreatic transformation. Importantly, we found a significant correlation among reduced HIPK2 expression, high grade of malignancy, and high nuclear size, a marker of increased ploidy. Overall, these results indicate that HIPK2 acts as a caretaker gene, whose inactivation increases tumorigenicity and causes CIN by cytokinesis failure.

No MeSH data available.


Related in: MedlinePlus

CIN and aneuploidy in E1A/Ras MEFsA, Hipk2+/+ and −/− MEFs were fixed at indicated passages (p) after stable transfection, stained with Hoechst and anti-beta-Tubulin-Cy3 Ab to identify the nuclei and the cytoplasm, respectively. About 1,000 cells per sample were scored for the presence of one or two nuclei/cell and the data are represented as mean ± SD (*P <0.05, Student t test). B, DNA content analysis of E1A/Ras MEFs at p12 after stable transfection. Dashed lines outline 2N and 4N DNA content. C, Representative images of Hoechst-stained metaphase spreads of indicated MEFs are shown; scale bar, 10 ξm. Chromosome number is reported in each image. D, The percentage of metaphases with the indicated chromosome number is shown; at least 65 metaphases were analyzed for each sample.
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Figure 2: CIN and aneuploidy in E1A/Ras MEFsA, Hipk2+/+ and −/− MEFs were fixed at indicated passages (p) after stable transfection, stained with Hoechst and anti-beta-Tubulin-Cy3 Ab to identify the nuclei and the cytoplasm, respectively. About 1,000 cells per sample were scored for the presence of one or two nuclei/cell and the data are represented as mean ± SD (*P <0.05, Student t test). B, DNA content analysis of E1A/Ras MEFs at p12 after stable transfection. Dashed lines outline 2N and 4N DNA content. C, Representative images of Hoechst-stained metaphase spreads of indicated MEFs are shown; scale bar, 10 ξm. Chromosome number is reported in each image. D, The percentage of metaphases with the indicated chromosome number is shown; at least 65 metaphases were analyzed for each sample.

Mentions: To investigate the occurrence of CIN after HIPK2-dependent cytokinesis failure, we measured the frequency of binucleated cells that accumulate during the passages of asynchronously growing MEFs. The morphological evaluation of adherent MEFs was assessed after tubulin immunostaining (Figure 2A). A higher frequency of binucleated cells was observed in the E1A/Ras Hipk2−/− MEFs compared with the Hipk2+/+ counterparts at early passage after stable transfections. The fraction of binucleated cells increased with passages only in E1A/Ras Hipk2−/− MEFs, suggesting that a process of CIN was present after cytokinesis failure due to the hipk2 absence (Figure 2A). At late passages after stable transfection, we also analyzed DNA content of the E1A/Ras MEFs by cytofluorimetric analysis. A strong reduction of the diploid population with a shift towards cells with a double DNA content and a broad population of cells with DNA content >4N, rather than the appearance of a distinct peak of 8N cells, suggest the occurrence of near-tetraploid cells in the E1A/Ras Hipk2−/− MEFs (Figure 2B).


HIPK2 deficiency causes chromosomal instability by cytokinesis failure and increases tumorigenicity.

Valente D, Bossi G, Moncada A, Tornincasa M, Indelicato S, Piscuoglio S, Karamitopoulou ED, Bartolazzi A, Pierantoni GM, Fusco A, Soddu S, Rinaldo C - Oncotarget (2015)

CIN and aneuploidy in E1A/Ras MEFsA, Hipk2+/+ and −/− MEFs were fixed at indicated passages (p) after stable transfection, stained with Hoechst and anti-beta-Tubulin-Cy3 Ab to identify the nuclei and the cytoplasm, respectively. About 1,000 cells per sample were scored for the presence of one or two nuclei/cell and the data are represented as mean ± SD (*P <0.05, Student t test). B, DNA content analysis of E1A/Ras MEFs at p12 after stable transfection. Dashed lines outline 2N and 4N DNA content. C, Representative images of Hoechst-stained metaphase spreads of indicated MEFs are shown; scale bar, 10 ξm. Chromosome number is reported in each image. D, The percentage of metaphases with the indicated chromosome number is shown; at least 65 metaphases were analyzed for each sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496358&req=5

Figure 2: CIN and aneuploidy in E1A/Ras MEFsA, Hipk2+/+ and −/− MEFs were fixed at indicated passages (p) after stable transfection, stained with Hoechst and anti-beta-Tubulin-Cy3 Ab to identify the nuclei and the cytoplasm, respectively. About 1,000 cells per sample were scored for the presence of one or two nuclei/cell and the data are represented as mean ± SD (*P <0.05, Student t test). B, DNA content analysis of E1A/Ras MEFs at p12 after stable transfection. Dashed lines outline 2N and 4N DNA content. C, Representative images of Hoechst-stained metaphase spreads of indicated MEFs are shown; scale bar, 10 ξm. Chromosome number is reported in each image. D, The percentage of metaphases with the indicated chromosome number is shown; at least 65 metaphases were analyzed for each sample.
Mentions: To investigate the occurrence of CIN after HIPK2-dependent cytokinesis failure, we measured the frequency of binucleated cells that accumulate during the passages of asynchronously growing MEFs. The morphological evaluation of adherent MEFs was assessed after tubulin immunostaining (Figure 2A). A higher frequency of binucleated cells was observed in the E1A/Ras Hipk2−/− MEFs compared with the Hipk2+/+ counterparts at early passage after stable transfections. The fraction of binucleated cells increased with passages only in E1A/Ras Hipk2−/− MEFs, suggesting that a process of CIN was present after cytokinesis failure due to the hipk2 absence (Figure 2A). At late passages after stable transfection, we also analyzed DNA content of the E1A/Ras MEFs by cytofluorimetric analysis. A strong reduction of the diploid population with a shift towards cells with a double DNA content and a broad population of cells with DNA content >4N, rather than the appearance of a distinct peak of 8N cells, suggest the occurrence of near-tetraploid cells in the E1A/Ras Hipk2−/− MEFs (Figure 2B).

Bottom Line: In particular, HIPK2 is required to complete cytokinesis and impaired HIPK2 expression results in cytokinesis failure and tetraploidization.Importantly, we found a significant correlation among reduced HIPK2 expression, high grade of malignancy, and high nuclear size, a marker of increased ploidy.Overall, these results indicate that HIPK2 acts as a caretaker gene, whose inactivation increases tumorigenicity and causes CIN by cytokinesis failure.

View Article: PubMed Central - PubMed

Affiliation: Experimental Oncology Laboratory, Regina Elena National Cancer Institute, Rome, Italy.

ABSTRACT
HIPK2, a cell fate decision kinase inactivated in several human cancers, is thought to exert its oncosuppressing activity through its p53-dependent and -independent apoptotic function. However, a HIPK2 role in cell proliferation has also been described. In particular, HIPK2 is required to complete cytokinesis and impaired HIPK2 expression results in cytokinesis failure and tetraploidization. Since tetraploidy may yield to aneuploidy and chromosomal instability (CIN), we asked whether unscheduled tetraploidy caused by loss of HIPK2 might contribute to tumorigenicity. Here, we show that, compared to Hipk2+/+ mouse embryo fibroblasts (MEFs), hipk2- MEFs accumulate subtetraploid karyotypes and develop CIN. Accumulation of these defects inhibits proliferation and spontaneous immortalization of primary MEFs whereas increases tumorigenicity when MEFs are transformed by E1A and Harvey-Ras oncogenes. Upon mouse injection, E1A/Ras-transformed hipk2- MEFs generate tumors with genetic alterations resembling those of human cancers derived by initial tetraploidization events, such as pancreatic adenocarcinoma. Thus, we evaluated HIPK2 expression in different stages of pancreatic transformation. Importantly, we found a significant correlation among reduced HIPK2 expression, high grade of malignancy, and high nuclear size, a marker of increased ploidy. Overall, these results indicate that HIPK2 acts as a caretaker gene, whose inactivation increases tumorigenicity and causes CIN by cytokinesis failure.

No MeSH data available.


Related in: MedlinePlus