Reduction in mortalin level by its antisense expression causes senescence-like growth arrest in human immortalized cells.
BACKGROUND
Human normal cells have active p53 and pRB tumor suppressor pathways and undergo telomere shortening at each cell division. When immortalized, these exhibit functional inactivation of one or both tumor suppressor pathways and activation of telomere-maintaining mechanisms. Regulation of immortalization promoting molecular pathways by other genes is poorly understood and is an essential component of cancer therapeutics. In the present study, we have immortalized human normal cells by functional inactivation of p53 and pRB tumor suppressor proteins and simultaneous activation of telomerase function. We demonstrate that when the expression of mortalin (a member of hsp70 family proteins) was suppressed in such genetically defined immortal cells they underwent a permanent growth arrest.


METHODS
WI-38 cells were transfected with expression plasmids encoding papilloma virus E6 and E7 proteins and a catalytic subunit of human telomerase enzyme. The derivative cells were compromised for p53 and pRB senescence pathways, showed telomerase activity and were immortalized. These cells were transfected with antisense expression plasmid for mortalin. The derivative clones were analyzed for mortalin expression, proliferation and telomerase activity.


RESULTS
WI-38 cells were immortalized with E6, E7 and the catalytic subunit of human telomerase. The derivative-immortalized cells when suppressed for mortalin expression underwent senescence-like growth arrest.


CONCLUSIONS
The data demonstrate that suppression of mortalin is sufficient to induce growth arrest in human immortalized cells that have compromised p53 and pRB functions and possess telomerase activity.
Wadhwa
Renu
R
National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305- 8562, Japan.
Takano
Syuichi
S
Taira
Kazunari
K
Kaul
Sunil C
SC
D016375:Antisense Elements (Genetics) / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002461:Cell Line, Transformed; D016922:Cellular Senescence / Q000235:genetics*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D024101:Mitochondrial Proteins; D009856:Oncogene Proteins, Viral / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D010957:Plasmids / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D019098:Telomerase / Q000235:genetics; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D016375:Antisense Elements (Genetics); C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; D018840:HSP70 Heat-Shock Proteins; C492001:HSPA9 protein, human; D024101:Mitochondrial Proteins; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; C080352:mortalin; C059731:oncogene protein E7, Human papillomavirus type 16; D019098:Telomerase