Frequent occurrence of protein isoforms with or without a single amino acid residue by subtle alternative splicing: the case of Gln in DRPLA affects subcellular localization of the products.

Protein isoforms with or without a single amino acid residue make a subtle difference. It has been documented on a few genes that alternative splicing generated such isoforms; however, the fact has attracted little attention. We became aware of a subtle sequence difference in DRPLA, a polyglutamine disease gene for dentatorubral pallidoluysian atrophy. Some reported cDNA sequences lacked 3 nucleotides (nt) (CAG), which were positioned apart from the expandable and polymorphic CAG repeats and also coded for glutamine. We experimentally confirmed that the difference was indeed generated by alternative splicing utilizing two acceptors separated by 3 nt. In DRPLA, the expression ratio of two mRNA isoforms was almost constant among tissues, with the CAG-included form being major. The glutamine-included protein isoform was more predominantly localized in the nucleus. Database searching revealed that alternative splice acceptors, as well as donors, are frequently situated very close to each other. We experimentally confirmed two mRNA isoforms of 3 nt difference in more than 200 cases by RT-PCR and found interesting features associated with this phenomena. Inclusion of 3 nt tends to result in single amino acid inclusion despite the phase of translational frame. The expression ratio sometimes varied extensively among tissues.

Nutritional deficiency affects cell cycle status and viability in A549 cells: role of p27Kip1.

We investigated how nutritional deficiency affects cell cycle and cell viability in A549 lung adenocarcinoma cells. Deprivation of various amino acids or glucose induced cell cycle arrest and cell death in a different manner. Cell death on deprivation of these nutrients was increased by downregulating of p27Kip1 with RNA interference. It was also observed that intrinsic p27Kip1 was segregated in cytoplasm in a glucose-deprived situation. In conclusion, amino acid or glucose deprivation induced cell cycle arrest and cell death, part of which is thought to be rescued by the existence of cytoplasmic p27Kip1.

A methylated oligonucleotide induced methylation of GSTP1 promoter and suppressed its expression in A549 lung adenocarcinoma cells.

Glutathione S-transferase P1 (GSTP1) belongs to xenobiotic enzymes, and is supposed to contribute to chemoresistance. Though it was reported that GSTP1 gene is suppressed by cytosine-guanine (CpG) island methylation of its promoter, this promoter is not strongly methylated and GSTP1 protein is highly expressed in lung cancer. We intended to induce methylation of GSTP1 CpG island by using a methylated sense oligonucleotide complementary to this region. When we transduced the methylated oligonucleotides to A549 lung adenocarcinoma cells, methylation of the GSTP1 promoter and reduction of GSTP1 expression was induced, cell viability was reduced; however, chemoresistance against cisplatin has not clearly changed.

Glutathione S-transferase P1 has protective effects on cell viability against camptothecin.

Glutathione S-transferase P1 (GSTP1) is one of the important xenobiotic-metabolizing enzymes. It was reported that GSTP1 was overexpressed in malignant tissues, and its expression level was associated with resistance to chemotherapeutics. We carried out transfection of GSTP1 sense and antisense vectors to examine effects of GSTP1 on cell cycle arrest and apoptosis induced by camptothecin in HeLa cells. Transfection of GSTP1 antisense vector induced apoptosis. Camptothecin-induced S- or G2/M arrest was intensified by transfection of GSTP1 antisense vector, and subsequent apoptosis was attenuated by transfection of GSTP1 sense vector. These results suggest that GSTP1 has protective effects against camptothecin-induced cytotoxicity.

Depletion of glutathione S-transferase P1 induces apoptosis in human lung fibroblasts.

Glutathione S-transferase P1 (GSTP1) is one of the xenobiotic-metabolizing and antioxidant enzymes, identified in the peripheral lungs. Recently, the authors reported the association between GSTP1 gene polymorphism and susceptibility to chronic obstructive pulmonary disease (COPD), and protective effect of GSTP1 against cigarette smoke in human lung fibroblasts in vitro. In this study, the authors investigated that depletion of GSTP1 by itself could induce cell death, including apoptosis, in human lung fibroblast-derived HFL-1 cells. The level of apoptosis and necrosis was increased significantly with GSTP1 antisense vector transfection. It was also observed that the transfection efficiency and the expression level of the vector were weaker in the transfectant of the antisense vector than in those of the sense and control vectors, which is also thought to indicate that inhibition of GSTP1 expression by the antisense vector alone affects cellular viability. However, there was no difference among these transfectants neither on glutathione (GSH) level nor on c-Jun NH2-terminal kinase (JNK) activation. Therefore, the authors report here that underexpression of GSTP1 appeared to induce apoptosis on lung fibroblasts, which suggests that GSTP1 may have protective effects against apoptosis in the airway cells, though the mechanism of this apoptotic pathway is still to be elucidated.