Abstract
GL331 is a novel podophyllotoxin-derived compound. In this study, GL331 induced human lung adenocarcinoma cell line CL1-5 growth arrest before death during the initial 24-h incubation period. We found that GL331 had no inhibitory effect on the expression of cyclins E, A, B1, CDK 4, and CDK 2; instead, its cell growth-inhibitory effect was partly attributable to an early down-regulation of cyclin D1 expression and in turn the reduction of retinoblastoma protein phosphorylation. GL331 enhanced the proteolysis of cyclin D1, and a proteasome inhibitor was able to block GL331-caused cyclin D1 reduction, suggesting that GL331-stimulated cyclin D1 degradation was through proteasomal processes. Additionally, GL331 reduced cellular cyclin D1 mRNA level down to 45% of control in 4 h and further to around 20% in 12 h. However, GL331 did not accelerate the disappearance of cyclin D1 mRNA under the condition of transcription blockage induced by actinomycin D. It was reported that a certain region in the 3′-untranslated region (UTR) of cyclin D1 mRNA mediated the mRNA degradation upon extracellular stresses. Herein, transient transfection studies demonstrated that the 3′-UTR insertion did not confer the susceptibility of luciferase reporter gene to the GL331 treatment. Together, these data suggested that GL331 did not decrease the stability of cyclin D1 mRNA. On the other hand, we found that GL331 specifically inhibited the cyclin D1 promoter-driven luciferase reporter activity. Western blot analyses showed that GL331 decreased the level of phosphorylated extracellular signal-regulated kinase (Erk), with no effect on p38 or c-Jun NH2-terminal kinase. Furthermore, GL331's inhibition of cyclin D1 promoter was attenuated by ectopic Erk-2 overexpression. These data suggested that GL331 inhibited cyclin D1 gene transcription via the Erk signaling pathway. In summary, we report that GL331 induced an early decline of cyclin D1 expression by dual mechanisms: 1) enhancement of protein turnover and 2) repression of Erk-mediated gene transcription.
Footnotes
-
↵1 Current address: Central Laboratory, Shin Kong Memorial Hospital, Taipei 111, Taiwan, Republic of China.
- Abbreviations:
- CDK
- cyclin-dependent kinase
- Rb
- retinoblastoma protein
- MAPK
- mitogen-activated protein kinase
- Erk
- extracellular signal-regulated kinase
- PGA2
- prostaglandin A2
- UTR
- untranslated region
- Topo II
- topoisomerase II
- CDC
- cell division cycle protein
- JNK
- Jun NH2-terminal kinase
- PBS
- phosphate-buffered saline
- PBST
- phosphate-buffered saline-Tween 20
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- N-CBZ-L-L-L-AL
- N-carbobenzyloxy-leucine-leucine-leucine-aldehyde
- Received March 5, 2001.
- Accepted June 18, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|