Limits...
Chromosome 6 encoded RNaseT2 protein is a cell growth regulator.

Liu J, Zhawar VK, Kaur G, Kaur GP, Deriel JK, Kandpal RP, Athwal RS - J. Cell. Mol. Med. (2009)

Bottom Line: A significant reduction was observed in colony forming efficiency, anchorage independence and growth rate of cells transfected with RNaseT2.The levels of transcripts involved in Akt signalling pathway, cell cycle control and pathways related to cell proliferation decreased 2-10-folds in SV40 immortalized cells in response to RNaseT2 expression.Our results are consistent with the conclusion that RNaseT2 is a cell growth regulator and it does not induce senescence in SV40 immortalized cell lines.

View Article: PubMed Central - PubMed

Affiliation: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA.

ABSTRACT
We have previously shown by chromosome transfer technique that chromosome 6 alters the phenotype of a variety of tumour cells and SV40 immortalized cells. We present here the phenotypic effects of the ectopic expression of RNaseT2, a highly conserved ribonuclease encoded by chromosome 6q27, in SV40 immortalized cell lines. We contrast our findings with those reported for ovarian carcinoma cell lines and an SV40 immortalized cell line transfected with RNaseT2. Although RNaseT2 expression is elevated in normal diploid fibroblasts approaching senescence (passage 64), forced expression of the gene in immortalized cells does not cause them to senesce. A significant reduction was observed in colony forming efficiency, anchorage independence and growth rate of cells transfected with RNaseT2. The levels of transcripts involved in Akt signalling pathway, cell cycle control and pathways related to cell proliferation decreased 2-10-folds in SV40 immortalized cells in response to RNaseT2 expression. Interestingly, some immortalized cells expressed alternatively spliced transcript variants instead of the full-length RNaseT2 transcript. Our results are consistent with the conclusion that RNaseT2 is a cell growth regulator and it does not induce senescence in SV40 immortalized cell lines.

Show MeSH

Related in: MedlinePlus

Effect of RNaseT2 overexpression on the growth of SV40 immortalized human epithelial CRL-9609 and fibroblast CSG2 cells. (A) The multiplication of CRL-9609 cells, stably expressing RNaseT2, measured using MTT assay at different time-points. Each point in the curve represents the average of six replicates. Clones 7, 12 and 20 express FLAG-tagged RNaseT2 recombinant protein, and clone 2 is a negative transfer clone. (B) The growth of CSG2 cells, expressing RNaseT2 was measured as above. Clones 3, 9 and 13 express FLAG-tagged RNaseT2 recombinant protein, and clone 5 is a negative gene transfer clone.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3822751&req=5

fig05: Effect of RNaseT2 overexpression on the growth of SV40 immortalized human epithelial CRL-9609 and fibroblast CSG2 cells. (A) The multiplication of CRL-9609 cells, stably expressing RNaseT2, measured using MTT assay at different time-points. Each point in the curve represents the average of six replicates. Clones 7, 12 and 20 express FLAG-tagged RNaseT2 recombinant protein, and clone 2 is a negative transfer clone. (B) The growth of CSG2 cells, expressing RNaseT2 was measured as above. Clones 3, 9 and 13 express FLAG-tagged RNaseT2 recombinant protein, and clone 5 is a negative gene transfer clone.

Mentions: Western blot analysis revealed the expression of the fusion protein in slow-growing clones, but not in fast-growing clones (Fig. 4A and B). In situ visualization of the tagged RNaseT2, by immunofluorescence, showed uniform distribution throughout the cytoplasm with a lesser signal associated with the nucleus (Fig. 4C). Even after culturing for 6 months, five clones of CSG2/RNaseT2 and six clones of CRL-9609/RNaseT2 showed expression of RNaseT2 protein. The cells from these clones continued to multiply at an appreciably slower, but measurable rate, relative to the parental cells and vector control. While the growth rate of CRL-9609/RNaseT2 clones was slightly slower than the parent cell line (Fig. 5A), the growth of CSG2/RNaseT2 clones was reduced dramatically (Fig. 5B). Similar effects were also seen on cloning efficiency and anchorage-independent growth of cells expressing RNaseT2 (Figs 6 and 7). The cloning efficiency of parent CRL-9609 cells, vector transfected clone and a non-expressing transfected clone varied between 52% and 56% as compared to 17–18% observed for RNaseT2 expressing clones of CRL-9609 (Fig. 6, parts A and B). Similarly the colony forming efficiencies for CSG2 parental cells, a vector transfected clone and a non-expressing clone varied between 53% and 65%, but RNaseT2 expressing clones of CSG2 had a significantly reduced cloning efficiency that varied between 4% and 6% (Fig. 6, parts C and D). Moreover, the CSG2/RNaseT2 clones formed much smaller colonies relative to the CRL9609/RNaseT2 clones. In soft agar, CRL-9609 and CSG2 gave an average of approximately 2000 and 2500 colonies, respectively. However, RNaseT2 expressing clones of CRL-9609 formed an average of 18, 144 and 1059 colonies for three different clones, respectively. Three RNaseT2 expressing CSG2 clones, on the other hand, formed an average of 17, 27 and 316 colonies, respectively (Fig. 7). Thus the colony forming efficiency and the ability to grow in soft agar of RNaseT2 expressing clones was significantly reduced relative to the controls.


Chromosome 6 encoded RNaseT2 protein is a cell growth regulator.

Liu J, Zhawar VK, Kaur G, Kaur GP, Deriel JK, Kandpal RP, Athwal RS - J. Cell. Mol. Med. (2009)

Effect of RNaseT2 overexpression on the growth of SV40 immortalized human epithelial CRL-9609 and fibroblast CSG2 cells. (A) The multiplication of CRL-9609 cells, stably expressing RNaseT2, measured using MTT assay at different time-points. Each point in the curve represents the average of six replicates. Clones 7, 12 and 20 express FLAG-tagged RNaseT2 recombinant protein, and clone 2 is a negative transfer clone. (B) The growth of CSG2 cells, expressing RNaseT2 was measured as above. Clones 3, 9 and 13 express FLAG-tagged RNaseT2 recombinant protein, and clone 5 is a negative gene transfer clone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822751&req=5

fig05: Effect of RNaseT2 overexpression on the growth of SV40 immortalized human epithelial CRL-9609 and fibroblast CSG2 cells. (A) The multiplication of CRL-9609 cells, stably expressing RNaseT2, measured using MTT assay at different time-points. Each point in the curve represents the average of six replicates. Clones 7, 12 and 20 express FLAG-tagged RNaseT2 recombinant protein, and clone 2 is a negative transfer clone. (B) The growth of CSG2 cells, expressing RNaseT2 was measured as above. Clones 3, 9 and 13 express FLAG-tagged RNaseT2 recombinant protein, and clone 5 is a negative gene transfer clone.
Mentions: Western blot analysis revealed the expression of the fusion protein in slow-growing clones, but not in fast-growing clones (Fig. 4A and B). In situ visualization of the tagged RNaseT2, by immunofluorescence, showed uniform distribution throughout the cytoplasm with a lesser signal associated with the nucleus (Fig. 4C). Even after culturing for 6 months, five clones of CSG2/RNaseT2 and six clones of CRL-9609/RNaseT2 showed expression of RNaseT2 protein. The cells from these clones continued to multiply at an appreciably slower, but measurable rate, relative to the parental cells and vector control. While the growth rate of CRL-9609/RNaseT2 clones was slightly slower than the parent cell line (Fig. 5A), the growth of CSG2/RNaseT2 clones was reduced dramatically (Fig. 5B). Similar effects were also seen on cloning efficiency and anchorage-independent growth of cells expressing RNaseT2 (Figs 6 and 7). The cloning efficiency of parent CRL-9609 cells, vector transfected clone and a non-expressing transfected clone varied between 52% and 56% as compared to 17–18% observed for RNaseT2 expressing clones of CRL-9609 (Fig. 6, parts A and B). Similarly the colony forming efficiencies for CSG2 parental cells, a vector transfected clone and a non-expressing clone varied between 53% and 65%, but RNaseT2 expressing clones of CSG2 had a significantly reduced cloning efficiency that varied between 4% and 6% (Fig. 6, parts C and D). Moreover, the CSG2/RNaseT2 clones formed much smaller colonies relative to the CRL9609/RNaseT2 clones. In soft agar, CRL-9609 and CSG2 gave an average of approximately 2000 and 2500 colonies, respectively. However, RNaseT2 expressing clones of CRL-9609 formed an average of 18, 144 and 1059 colonies for three different clones, respectively. Three RNaseT2 expressing CSG2 clones, on the other hand, formed an average of 17, 27 and 316 colonies, respectively (Fig. 7). Thus the colony forming efficiency and the ability to grow in soft agar of RNaseT2 expressing clones was significantly reduced relative to the controls.

Bottom Line: A significant reduction was observed in colony forming efficiency, anchorage independence and growth rate of cells transfected with RNaseT2.The levels of transcripts involved in Akt signalling pathway, cell cycle control and pathways related to cell proliferation decreased 2-10-folds in SV40 immortalized cells in response to RNaseT2 expression.Our results are consistent with the conclusion that RNaseT2 is a cell growth regulator and it does not induce senescence in SV40 immortalized cell lines.

View Article: PubMed Central - PubMed

Affiliation: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA.

ABSTRACT
We have previously shown by chromosome transfer technique that chromosome 6 alters the phenotype of a variety of tumour cells and SV40 immortalized cells. We present here the phenotypic effects of the ectopic expression of RNaseT2, a highly conserved ribonuclease encoded by chromosome 6q27, in SV40 immortalized cell lines. We contrast our findings with those reported for ovarian carcinoma cell lines and an SV40 immortalized cell line transfected with RNaseT2. Although RNaseT2 expression is elevated in normal diploid fibroblasts approaching senescence (passage 64), forced expression of the gene in immortalized cells does not cause them to senesce. A significant reduction was observed in colony forming efficiency, anchorage independence and growth rate of cells transfected with RNaseT2. The levels of transcripts involved in Akt signalling pathway, cell cycle control and pathways related to cell proliferation decreased 2-10-folds in SV40 immortalized cells in response to RNaseT2 expression. Interestingly, some immortalized cells expressed alternatively spliced transcript variants instead of the full-length RNaseT2 transcript. Our results are consistent with the conclusion that RNaseT2 is a cell growth regulator and it does not induce senescence in SV40 immortalized cell lines.

Show MeSH
Related in: MedlinePlus