Limits...
Hyperstable U1snRNA complementary to the K-ras transcripts induces cell death in pancreatic cancer cells.

Kato K, Hitomi Y, Imamura K, Esumi H - Br. J. Cancer (2002)

Bottom Line: In this study, we utilised U1 small nuclear RNA (snRNA) that binds physiologically to the 5' splice site (5'ss) of pre-mRNA, to develop a novel vector system that permits imposed binding of antisense RNA to its target.This revealed that two of the hyperstable U1snRNAs induced cell death after gene transduction, and significantly reduced the number of G418-resistant colonies to less than 10% of the controls.Hyperstable U1snRNA might be a novel approach to express effective antisense RNA in target cells.

View Article: PubMed Central - PubMed

Affiliation: Investigative Treatment Division, National Cancer Center Research Institute East, 6-5-1, Kashiwanoha, Kashiwa, Chiba 277-8577, Japan.

Show MeSH

Related in: MedlinePlus

(A) Western blot analysis of p21 K-ras proteins. Total cell lysates (100 μg), prepared from parent and stable transfectants at early passage, were subjected to 12% SDS–PAGE, transferred to a membrane, and blotted with K-ras p21-specific monoclonal antibody. (B) Expression of hyperstable U1snRNA in PANC-1. The cells were transfected with the hyperstable U1snRNA plasmids and selected with G418. Total RNA (3 μg) was separated on a 5% polyacrylamide/7 M urea gel, blotted on a membrane, and hybridised with radiolabelled sense (upper panels) or antisense (lower panels) K-ras oligonucleotides corresponding to the target sites r1 to r5 (see Figure 1B). The position of antisense hyperstable U1snRNA (aU1), sense hyperstable U1snRNA (sU1), and wild-type U1snRNA (wtU1, 164 bases) is indicated. Hybridisation with U2B verified the integrity and load of RNA.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2376165&req=5

fig4: (A) Western blot analysis of p21 K-ras proteins. Total cell lysates (100 μg), prepared from parent and stable transfectants at early passage, were subjected to 12% SDS–PAGE, transferred to a membrane, and blotted with K-ras p21-specific monoclonal antibody. (B) Expression of hyperstable U1snRNA in PANC-1. The cells were transfected with the hyperstable U1snRNA plasmids and selected with G418. Total RNA (3 μg) was separated on a 5% polyacrylamide/7 M urea gel, blotted on a membrane, and hybridised with radiolabelled sense (upper panels) or antisense (lower panels) K-ras oligonucleotides corresponding to the target sites r1 to r5 (see Figure 1B). The position of antisense hyperstable U1snRNA (aU1), sense hyperstable U1snRNA (sU1), and wild-type U1snRNA (wtU1, 164 bases) is indicated. Hybridisation with U2B verified the integrity and load of RNA.

Mentions: Cell morphology of PANC-1 clones transfected with hyperstable U1snRNA. Parent PANC-1 (A) and transfectants, Pr1A-4 (B), Pr1S (C), Pr3A-4 (D), Pr3A-5 (E), and Pr3S (F) were cultured to confluence and photographed at a magnification of ×200. The r1A and r3A transfected clones are heterogeneous in their cell morphology and contain many multinucleated giant cells.


Hyperstable U1snRNA complementary to the K-ras transcripts induces cell death in pancreatic cancer cells.

Kato K, Hitomi Y, Imamura K, Esumi H - Br. J. Cancer (2002)

(A) Western blot analysis of p21 K-ras proteins. Total cell lysates (100 μg), prepared from parent and stable transfectants at early passage, were subjected to 12% SDS–PAGE, transferred to a membrane, and blotted with K-ras p21-specific monoclonal antibody. (B) Expression of hyperstable U1snRNA in PANC-1. The cells were transfected with the hyperstable U1snRNA plasmids and selected with G418. Total RNA (3 μg) was separated on a 5% polyacrylamide/7 M urea gel, blotted on a membrane, and hybridised with radiolabelled sense (upper panels) or antisense (lower panels) K-ras oligonucleotides corresponding to the target sites r1 to r5 (see Figure 1B). The position of antisense hyperstable U1snRNA (aU1), sense hyperstable U1snRNA (sU1), and wild-type U1snRNA (wtU1, 164 bases) is indicated. Hybridisation with U2B verified the integrity and load of RNA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376165&req=5

fig4: (A) Western blot analysis of p21 K-ras proteins. Total cell lysates (100 μg), prepared from parent and stable transfectants at early passage, were subjected to 12% SDS–PAGE, transferred to a membrane, and blotted with K-ras p21-specific monoclonal antibody. (B) Expression of hyperstable U1snRNA in PANC-1. The cells were transfected with the hyperstable U1snRNA plasmids and selected with G418. Total RNA (3 μg) was separated on a 5% polyacrylamide/7 M urea gel, blotted on a membrane, and hybridised with radiolabelled sense (upper panels) or antisense (lower panels) K-ras oligonucleotides corresponding to the target sites r1 to r5 (see Figure 1B). The position of antisense hyperstable U1snRNA (aU1), sense hyperstable U1snRNA (sU1), and wild-type U1snRNA (wtU1, 164 bases) is indicated. Hybridisation with U2B verified the integrity and load of RNA.
Mentions: Cell morphology of PANC-1 clones transfected with hyperstable U1snRNA. Parent PANC-1 (A) and transfectants, Pr1A-4 (B), Pr1S (C), Pr3A-4 (D), Pr3A-5 (E), and Pr3S (F) were cultured to confluence and photographed at a magnification of ×200. The r1A and r3A transfected clones are heterogeneous in their cell morphology and contain many multinucleated giant cells.

Bottom Line: In this study, we utilised U1 small nuclear RNA (snRNA) that binds physiologically to the 5' splice site (5'ss) of pre-mRNA, to develop a novel vector system that permits imposed binding of antisense RNA to its target.This revealed that two of the hyperstable U1snRNAs induced cell death after gene transduction, and significantly reduced the number of G418-resistant colonies to less than 10% of the controls.Hyperstable U1snRNA might be a novel approach to express effective antisense RNA in target cells.

View Article: PubMed Central - PubMed

Affiliation: Investigative Treatment Division, National Cancer Center Research Institute East, 6-5-1, Kashiwanoha, Kashiwa, Chiba 277-8577, Japan.

Show MeSH
Related in: MedlinePlus