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Par-3 partitioning defective 3 homolog (C. elegans) and androgen-induced prostate proliferative shutoff associated protein genes are mutationally inactivated in prostate cancer cells.

Kunnev D, Ivanov I, Ionov Y - BMC Cancer (2009)

Bottom Line: Sequencing identified inactivating mutations in both alleles of the PARD3 and AS3 genes in the LNCaP and 22RV1 cells, respectively.Introduction of a wild-type PARD3 cDNA into the LNCaP cells resulted in a higher proliferation rate in tissue culture, a higher adhesion of LNCaP cells to the components of extracellular matrix and impaired the growth of the LNCaP cells in soft agar and in a three-dimensional cell-culture.The mutational inactivation in a prostate cancer cell line of the PARD3 gene involved in asymmetric cell division and maintenance of cell-polarity suggests that the loss of cell-polarity contributes to prostate carcinogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. Dimiter.Kunnev@Roswellpark.org

ABSTRACT

Background: Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven its usefulness in identifying mutant genes in cancer cell lines. An increase in transcription in response to NMD inhibition of a subset of genes is a major cause of false positives when genes are selected for sequencing analysis. To distinguish between mRNA accumulations caused by stress response-induced transcription and nonsense-containing mRNA stabilizations is a challenge in identifying mutant genes using GINI.

Methods: To identify potential tumor-suppressor genes mutated in prostate cancer cell lines, we applied a version of GINI that involves inhibition of NMD in two steps. In the first step, NMD is inhibited in duplicate tissue-culture plates. During this step, both the substrate for NMD and stress-response mRNA transcripts are accumulated in cells. In the second step, transcription is inhibited in both plates and NMD is inhibited in one plate and released in the second plate. Microarray analysis of gene-expression profiles in both plates after the second step detects only the differences in mRNA degradation but not in mRNA accumulation.

Results: Analyzing gene expression profile alterations in 22RV1 and LNCaP prostate cancer cells following NMD inhibition we selected candidates for sequencing analysis in both cell lines. Sequencing identified inactivating mutations in both alleles of the PARD3 and AS3 genes in the LNCaP and 22RV1 cells, respectively. Introduction of a wild-type PARD3 cDNA into the LNCaP cells resulted in a higher proliferation rate in tissue culture, a higher adhesion of LNCaP cells to the components of extracellular matrix and impaired the growth of the LNCaP cells in soft agar and in a three-dimensional cell-culture.

Conclusion: The mutational inactivation in a prostate cancer cell line of the PARD3 gene involved in asymmetric cell division and maintenance of cell-polarity suggests that the loss of cell-polarity contributes to prostate carcinogenesis.

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Effect of Par3 protein expression on cell morphology, focal adhesion contacts, cell-cell contacts and proliferation rate. (A). Phase contrast images (200×) of the LNCaP cells expressing or not expressing Par3 protein. (B). Immunoflurescent images showing the foci of vinculin (vin) - representative marker for focal adhesion contacts (stained with red indicated with arrows), E-cadherin - (E-cad) representative marker for adherens junctions (stained with red). Zonula occludens-1 (ZO-1) - representative marker for tight junctions is presented with overlay pictures from three independent layers: Differential Interference Contrast (DIC) visualizing cells shape, ZO-1 stained with red (indicated with arrows) and nuclei stained with blue. Size bar is 20 μm. (C). 5 × 103 cells per well were seeded in 24-well format tissue culture plate in RPMI 1640 containing 5% fetal calf serum (FBS). Cells were cultured for indicated time periods, harvested and counted by hemocytometer. Cell numbers are shown as the average ± S.D. of counting of cells in four wells per time-point. The experiment was repeated independently three times with similar results.
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Figure 3: Effect of Par3 protein expression on cell morphology, focal adhesion contacts, cell-cell contacts and proliferation rate. (A). Phase contrast images (200×) of the LNCaP cells expressing or not expressing Par3 protein. (B). Immunoflurescent images showing the foci of vinculin (vin) - representative marker for focal adhesion contacts (stained with red indicated with arrows), E-cadherin - (E-cad) representative marker for adherens junctions (stained with red). Zonula occludens-1 (ZO-1) - representative marker for tight junctions is presented with overlay pictures from three independent layers: Differential Interference Contrast (DIC) visualizing cells shape, ZO-1 stained with red (indicated with arrows) and nuclei stained with blue. Size bar is 20 μm. (C). 5 × 103 cells per well were seeded in 24-well format tissue culture plate in RPMI 1640 containing 5% fetal calf serum (FBS). Cells were cultured for indicated time periods, harvested and counted by hemocytometer. Cell numbers are shown as the average ± S.D. of counting of cells in four wells per time-point. The experiment was repeated independently three times with similar results.

Mentions: Examination with a light microscope did not detect significant differences in appearance between the LNCaP cells, ectopically expressing Par3 protein, and the LNCaP cells transfected with empty vector (Figure 3A). However, since slight alterations in cell shape still could be observed (Par3 expressing cells seem to look slightly more attached to the plastic) we investigated the results of ectopic Par3 expression on focal adhesion as well as on tight and on adherens junctions. Staining of control and Par3 expressing LNCaP cells with anti-vinculin antibodies has shown that focal adhesions was not affected by ectopic expression of Par3 (Figure 3B top). Neither had Par3 expression had an effect on adherens junction as shown by staining with anti E-cadherin antibody (Figure 3B middle). However, staining with anti-ZO-1 (used as a marker for tight junction) antibody has shown that tight junction might be affected by ectopic Par3 expression. Usually, the ZO-1 staining surrounds cells by continuous line when their tight junctions are well established. On the contrary, LNCaP cells are traced by ZO-1 staining with dots as a clear indication of not so well established tight junctions (Figure 3B EGFP bottom). The expression of Par3 increases partially the continuality of the ZO-1 signal which indicates some stabilization (but not restoring) of tight junctions (Figure 3B bottom).


Par-3 partitioning defective 3 homolog (C. elegans) and androgen-induced prostate proliferative shutoff associated protein genes are mutationally inactivated in prostate cancer cells.

Kunnev D, Ivanov I, Ionov Y - BMC Cancer (2009)

Effect of Par3 protein expression on cell morphology, focal adhesion contacts, cell-cell contacts and proliferation rate. (A). Phase contrast images (200×) of the LNCaP cells expressing or not expressing Par3 protein. (B). Immunoflurescent images showing the foci of vinculin (vin) - representative marker for focal adhesion contacts (stained with red indicated with arrows), E-cadherin - (E-cad) representative marker for adherens junctions (stained with red). Zonula occludens-1 (ZO-1) - representative marker for tight junctions is presented with overlay pictures from three independent layers: Differential Interference Contrast (DIC) visualizing cells shape, ZO-1 stained with red (indicated with arrows) and nuclei stained with blue. Size bar is 20 μm. (C). 5 × 103 cells per well were seeded in 24-well format tissue culture plate in RPMI 1640 containing 5% fetal calf serum (FBS). Cells were cultured for indicated time periods, harvested and counted by hemocytometer. Cell numbers are shown as the average ± S.D. of counting of cells in four wells per time-point. The experiment was repeated independently three times with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2749059&req=5

Figure 3: Effect of Par3 protein expression on cell morphology, focal adhesion contacts, cell-cell contacts and proliferation rate. (A). Phase contrast images (200×) of the LNCaP cells expressing or not expressing Par3 protein. (B). Immunoflurescent images showing the foci of vinculin (vin) - representative marker for focal adhesion contacts (stained with red indicated with arrows), E-cadherin - (E-cad) representative marker for adherens junctions (stained with red). Zonula occludens-1 (ZO-1) - representative marker for tight junctions is presented with overlay pictures from three independent layers: Differential Interference Contrast (DIC) visualizing cells shape, ZO-1 stained with red (indicated with arrows) and nuclei stained with blue. Size bar is 20 μm. (C). 5 × 103 cells per well were seeded in 24-well format tissue culture plate in RPMI 1640 containing 5% fetal calf serum (FBS). Cells were cultured for indicated time periods, harvested and counted by hemocytometer. Cell numbers are shown as the average ± S.D. of counting of cells in four wells per time-point. The experiment was repeated independently three times with similar results.
Mentions: Examination with a light microscope did not detect significant differences in appearance between the LNCaP cells, ectopically expressing Par3 protein, and the LNCaP cells transfected with empty vector (Figure 3A). However, since slight alterations in cell shape still could be observed (Par3 expressing cells seem to look slightly more attached to the plastic) we investigated the results of ectopic Par3 expression on focal adhesion as well as on tight and on adherens junctions. Staining of control and Par3 expressing LNCaP cells with anti-vinculin antibodies has shown that focal adhesions was not affected by ectopic expression of Par3 (Figure 3B top). Neither had Par3 expression had an effect on adherens junction as shown by staining with anti E-cadherin antibody (Figure 3B middle). However, staining with anti-ZO-1 (used as a marker for tight junction) antibody has shown that tight junction might be affected by ectopic Par3 expression. Usually, the ZO-1 staining surrounds cells by continuous line when their tight junctions are well established. On the contrary, LNCaP cells are traced by ZO-1 staining with dots as a clear indication of not so well established tight junctions (Figure 3B EGFP bottom). The expression of Par3 increases partially the continuality of the ZO-1 signal which indicates some stabilization (but not restoring) of tight junctions (Figure 3B bottom).

Bottom Line: Sequencing identified inactivating mutations in both alleles of the PARD3 and AS3 genes in the LNCaP and 22RV1 cells, respectively.Introduction of a wild-type PARD3 cDNA into the LNCaP cells resulted in a higher proliferation rate in tissue culture, a higher adhesion of LNCaP cells to the components of extracellular matrix and impaired the growth of the LNCaP cells in soft agar and in a three-dimensional cell-culture.The mutational inactivation in a prostate cancer cell line of the PARD3 gene involved in asymmetric cell division and maintenance of cell-polarity suggests that the loss of cell-polarity contributes to prostate carcinogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. Dimiter.Kunnev@Roswellpark.org

ABSTRACT

Background: Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven its usefulness in identifying mutant genes in cancer cell lines. An increase in transcription in response to NMD inhibition of a subset of genes is a major cause of false positives when genes are selected for sequencing analysis. To distinguish between mRNA accumulations caused by stress response-induced transcription and nonsense-containing mRNA stabilizations is a challenge in identifying mutant genes using GINI.

Methods: To identify potential tumor-suppressor genes mutated in prostate cancer cell lines, we applied a version of GINI that involves inhibition of NMD in two steps. In the first step, NMD is inhibited in duplicate tissue-culture plates. During this step, both the substrate for NMD and stress-response mRNA transcripts are accumulated in cells. In the second step, transcription is inhibited in both plates and NMD is inhibited in one plate and released in the second plate. Microarray analysis of gene-expression profiles in both plates after the second step detects only the differences in mRNA degradation but not in mRNA accumulation.

Results: Analyzing gene expression profile alterations in 22RV1 and LNCaP prostate cancer cells following NMD inhibition we selected candidates for sequencing analysis in both cell lines. Sequencing identified inactivating mutations in both alleles of the PARD3 and AS3 genes in the LNCaP and 22RV1 cells, respectively. Introduction of a wild-type PARD3 cDNA into the LNCaP cells resulted in a higher proliferation rate in tissue culture, a higher adhesion of LNCaP cells to the components of extracellular matrix and impaired the growth of the LNCaP cells in soft agar and in a three-dimensional cell-culture.

Conclusion: The mutational inactivation in a prostate cancer cell line of the PARD3 gene involved in asymmetric cell division and maintenance of cell-polarity suggests that the loss of cell-polarity contributes to prostate carcinogenesis.

Show MeSH
Related in: MedlinePlus