Growth-stimulating effects of tumor necrosis factor-alpha on simian virus 40-transformed human keratinocytes is linked to phosphorylation of retinoblastoma protein.
In this report, we show that tumor necrosis factor-alpha (TNF-alpha) has a strong growth-stimulating effect on simian virus 40 (SV40)-transformed keratinocytes, while it exerts a potent growth-inhibitory effect on normal human keratinocytes. Addition of TNF-alpha to SV40-transformed keratinocytes stimulated cell growth by 216, 246, or 248% at 25, 50, or 250 ng/ml after 4 days culture compared with untreated cells, while addition to normal human keratinocytes reduced growth to 65, 38, and 32% at the same concentrations, respectively. Cell cycle analysis by flow cytometry showed that treatment of SV40-transformed keratinocytes with 250 ng/ml TNF-alpha for 48 h increased the S-phase cells from 19.8 +/- 0.3 to 49.7 +/- 2.4%. The percentage of G1/G0 phase cells decreased from 62.6 +/- 1.6 to 32.4 +/- 3.3%. We also demonstrated by immunofluorescent staining that SV40 large T antigen was enhanced by TNF-alpha incubation. Additionally, we examined the hyper and hypo-phosphorylated state of retinoblastoma protein (pRB), the cell cycle regulatory protein. TNF-alpha induced hyperphosphorylated pRB in SV40-transformed keratinocytes. On the other hand, TNF-alpha suppressed it in normal keratinocytes. We hypothesize that the enhancement of SV40 large T antigen and hyperphosphorylated pRB is involved in the mechanism of growth stimulation of SV40-transformed keratinocytes by TNF-alpha.
Kobayashi
T
T
Department of Dermatology, Osaka University, School of Medicine, Suita, Japan.
Okumura
H
H
Hashimoto
K
K
Asada
H
H
Yoshikawa
K
K
D002455:Cell Division / Q000187:drug effects; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism* / Q000473:pathology*; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013539:Simian virus 40; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016160:Retinoblastoma Protein; D014409:Tumor Necrosis Factor-alpha