Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts

OBJECTIVE: Human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. The beneficial effects of vitamin E in aging have been established, but studies to determine the mechanisms of these effects are ongoing. This study determined the molecular mechanism of γ-tocotrienol, a vitamin E homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes. METHODS: Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer. RESULTS: The cell cycle was arrested in the G0/G1 phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with γ-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G0/G1 phase and increased cell populations in the G2/M phase. γ-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts. CONCLUSION: γ-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.

Molecular Characterization and Functional Prediction of a Novel Leaf SAG Encoding a CBS-domain-containing Protein from Coleus blumei / 中国生物化学与分子生物学报

Leaf senescence is considered as one of important factors to decrease ornamental values of foliage plants. In the attempt to study and understand the molecular mechanism of leaf senescence, a senescent leaf cDNA library of Coleus blumei was constructed and a small EST library was obtained. According to the sequence of an EST fragment with a cystathionine beta synthase (CBS) domain, a novel leaf senescenceassociated gene (SAG) full-length cDNA encoding a CBS-domain-containing protein, denoted Cbcbs, was rapidly cloned using a strategy of RACE combined with cDNA library. The full length of the Cbcbs gene was 859 bp long (accession No. EF076754) and contained a 609 bp open reading frame (ORF) encoding a 202amino acid protein. One stop codon (TAA) was found in 5' UTR and one possible polyadenylation signal,AATAAA, and a pentanucleotide motif, ATTTA, were found in 3' UTR. The CbCBS contained a predicted mitochondrial targeting peptide in the N-terminal region, two conserved and intact CBS domains, four casein kinase Ⅱ (CK Ⅱ) phosphorylation sites, three protein kinase c (PKC) phosphorylation sites and one tyrosine sulfation (TS) site. Sequence comparisons and phylogenetic analysis showed that CbCBS was a novel senescence or stress-associated protein. The prediction analysis of secondary structure and three dimensional structure of CbCBS suggested that the chief function of the protein was decided by the CBS domain pair. The expression pattern of Cbcbs in leaves was analyzed by RT-PCR. It was demonstrated that Cbcbs gene was a senescence-associated gene (SAG) and expressed in all leaf stages, young stage (Y) being the lowest and terminal senescence stage (S3) being the highest, and was upregulated along with the leaf senescence.Function analysis showed that the mature CbCBS maybe acts as a sensor of cellular energy status and directly or indirectly regulates cellular energy levels to increase ATP content in mitochondria during periods of metabolic stress of senescent leaves.