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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.

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Splice variants and gene structure of RNaseT2. (A) Aliquots of total RNA from human cell lines FS-2 (lane 1), GMO3468A (lane 2), AR5 (lane 3), GMO847 (lane 4), 6A-1–1 (lane 5), 6A-2–2 (lane 6), SKOV-3 (lane 7), A549 (lane 8) and U87MG3 (lane 9) were amplified by RT-PCR using primer pair F180/R588 that corresponds to Exon 1 and Exon 4. Lane 10 is a no-template control. The arrows indicate the amplified products of spliced variants 1 and 2. The figure is a composite of two gel pictures. (B) The boxes represent the relative positions of exons. The position of primer sequences that correspond to 5′ and 3′ ends of the gene are indicated by horizontal arrows. In-frame start codons are marked as ATG sequences and the vertical arrows indicate out-of-frame start codons.
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fig02: Splice variants and gene structure of RNaseT2. (A) Aliquots of total RNA from human cell lines FS-2 (lane 1), GMO3468A (lane 2), AR5 (lane 3), GMO847 (lane 4), 6A-1–1 (lane 5), 6A-2–2 (lane 6), SKOV-3 (lane 7), A549 (lane 8) and U87MG3 (lane 9) were amplified by RT-PCR using primer pair F180/R588 that corresponds to Exon 1 and Exon 4. Lane 10 is a no-template control. The arrows indicate the amplified products of spliced variants 1 and 2. The figure is a composite of two gel pictures. (B) The boxes represent the relative positions of exons. The position of primer sequences that correspond to 5′ and 3′ ends of the gene are indicated by horizontal arrows. In-frame start codons are marked as ATG sequences and the vertical arrows indicate out-of-frame start codons.

Mentions: The exon composition of transcripts observed in Northern blots was further investigated by exon-specific RT-PCR. Total RNA samples, isolated from normal diploid fibroblasts and variety of immortal cell lines, were amplified by using the primer pairs F180/R588 and F583/R1158. Variations in sizes of amplified products were detected at the 5′ end of the transcript (Fig. 2A) but not at the 3′ end (data not shown). The RT-PCR amplification of mRNA from normal diploid fibroblast cells with F180/R588 primers yielded a major 409 bp product (expected from the previously characterized 1.2 kb RNaseT2 mRNA) and a minor 268 bp product. While the 268 bp product was amplifiable from all cell lines tested, the 409 bp product was undetectable in A549 lung carcinoma, U87MG3 glioblastoma and in clones 6A-1–1 and 6A-2–2, the two immortal revertants of senescent chromosome 6 transfer clones. A 213 bp product was also detected in the RT-PCR products of mRNA isolated from clone 6A-1–1 and A549 lung carcinoma cell line. The sequencing of the 268 bp products of several cell lines revealed identical nucleotide sequences. Similarly, the sequences of the 213 bp product from RT-PCR of 6A-1–1 and A549 mRNAs also yielded identical sequences. The analyses of these sequences confirmed that the 268bp and 213 bp products are splice variants of RNaseT2.


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)

Splice variants and gene structure of RNaseT2. (A) Aliquots of total RNA from human cell lines FS-2 (lane 1), GMO3468A (lane 2), AR5 (lane 3), GMO847 (lane 4), 6A-1–1 (lane 5), 6A-2–2 (lane 6), SKOV-3 (lane 7), A549 (lane 8) and U87MG3 (lane 9) were amplified by RT-PCR using primer pair F180/R588 that corresponds to Exon 1 and Exon 4. Lane 10 is a no-template control. The arrows indicate the amplified products of spliced variants 1 and 2. The figure is a composite of two gel pictures. (B) The boxes represent the relative positions of exons. The position of primer sequences that correspond to 5′ and 3′ ends of the gene are indicated by horizontal arrows. In-frame start codons are marked as ATG sequences and the vertical arrows indicate out-of-frame start codons.
© Copyright Policy
Related In: Results  -  Collection

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

fig02: Splice variants and gene structure of RNaseT2. (A) Aliquots of total RNA from human cell lines FS-2 (lane 1), GMO3468A (lane 2), AR5 (lane 3), GMO847 (lane 4), 6A-1–1 (lane 5), 6A-2–2 (lane 6), SKOV-3 (lane 7), A549 (lane 8) and U87MG3 (lane 9) were amplified by RT-PCR using primer pair F180/R588 that corresponds to Exon 1 and Exon 4. Lane 10 is a no-template control. The arrows indicate the amplified products of spliced variants 1 and 2. The figure is a composite of two gel pictures. (B) The boxes represent the relative positions of exons. The position of primer sequences that correspond to 5′ and 3′ ends of the gene are indicated by horizontal arrows. In-frame start codons are marked as ATG sequences and the vertical arrows indicate out-of-frame start codons.
Mentions: The exon composition of transcripts observed in Northern blots was further investigated by exon-specific RT-PCR. Total RNA samples, isolated from normal diploid fibroblasts and variety of immortal cell lines, were amplified by using the primer pairs F180/R588 and F583/R1158. Variations in sizes of amplified products were detected at the 5′ end of the transcript (Fig. 2A) but not at the 3′ end (data not shown). The RT-PCR amplification of mRNA from normal diploid fibroblast cells with F180/R588 primers yielded a major 409 bp product (expected from the previously characterized 1.2 kb RNaseT2 mRNA) and a minor 268 bp product. While the 268 bp product was amplifiable from all cell lines tested, the 409 bp product was undetectable in A549 lung carcinoma, U87MG3 glioblastoma and in clones 6A-1–1 and 6A-2–2, the two immortal revertants of senescent chromosome 6 transfer clones. A 213 bp product was also detected in the RT-PCR products of mRNA isolated from clone 6A-1–1 and A549 lung carcinoma cell line. The sequencing of the 268 bp products of several cell lines revealed identical nucleotide sequences. Similarly, the sequences of the 213 bp product from RT-PCR of 6A-1–1 and A549 mRNAs also yielded identical sequences. The analyses of these sequences confirmed that the 268bp and 213 bp products are splice variants of RNaseT2.

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