Down regulation of the expression of the p110, p85 and p55 subunits of phosphatidylinositol 3-kinase during colon cancer cell anchorage-independent growth.
We have found that analogues of phosphatidylinositol modified at the 3'-myo-inositol position prevent phosphorylation by phosphatidylinositol 3-kinase (PI3-kinase) inhibit the anchorage-independent growth of HT-29 human colon adenocarcinoma cells as colonies in soft agarose but have no effect on monolayer growth on a plastic surface. Examination of an incomplete differential display library revealed 3 genes whose expression was increased and 11 genes whose expression was decreased or absent in HT-29 cells growing as colonies compared to monolayer growth. One of the cDNAs corresponding to an mRNA that was expressed only in cells growing as a monolayer was a 126 bp fragment that had 98% identity with a fragment of mRNA for human p55PIK PI3-kinase regulatory subunit. The down regulation of p55PIK gene expression in HT-29 cells growing as colonies was confirmed by RT-PCR using p55PIK-specific oligonucleotide primers. RT-PCR and Northern hybridisation were used to show that the expression of the p110 catalytic subunit and the p85 regulatory subunit of PI3-kinase were also down regulated in HT-29 cells growing as colonies. We measured a decrease of approximately 25% in total PI3-kinase activity of the HT-29 cells grown in soft agarose compared to HT-29 cells grown as monolayer. The results suggest that p110 PI3-kinase activity may be limiting in cells showing anchorage independent-growth which could explain their increased sensitivity, compared to cells in monolayer culture, to inhibitors of PI3-kinase signalling.
Yeates
L C
LC
Arizona Cancer Center, University of Arizona, Tucson 85724-5024, USA.
Gallegos
A
A
Kozikowski
A P
AP
Powis
G
G
D015152:Blotting, Northern; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D004587:Electrophoresis, Agar Gel; D015972:Gene Expression Regulation, Neoplastic; D019073:HT29 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D012333:RNA, Messenger; D019869:Phosphatidylinositol 3-Kinases