Isolation and characterization of the TIGA genes, whose transcripts are induced by growth arrest

We report here the isolation of 44 genes that are upregulated after serum starvation and/or contact inhibition. These genes have been termed TIGA, after Transcript Induced by Growth Arrest. We found that there are two kinds of G0 phases caused by serum starvation, namely, the shallow G0 (or G0/G1) and the deep G0 phases. The shallow G0 is induced by only a few hours of serum starvation, while deep G0 is generated after 3 days of serum starvation. We propose that mammalian cells enter deep G0 through a G0 gate, which is only opened on the third day of serum starvation. TIGA1, one of the uncharacterized TIGA genes, encodes a homolog of cyanate permease of bacteria and localizes in mitochondria. This suggests that Tiga1 is involved in the inorganic ion transport and metabolism needed to maintain the deep G0 phase. Ectopic expression of TIGA1 inhibited not only tumor cell proliferation but also anchorage-independent growth of cancer cell lines. A microsatellite marker, ENDL-1, allowed us to detect loss of heterozygosity around the TIGA1 gene region (5q21–22). Further analysis of the TIGA genes we have identified here may help us to better understand the mechanisms that regulate the G0 phase

Brother of the regulator of the imprinted site (BORIS) variant subfamily 6 is a novel target of lung cancer stem-like cell immunotherapy.

Lung cancer is one of the most common malignancies with a high rate of mortality. Lung cancer stem-like cells (CSCs)/ cancer-initiating cells (CICs) play major role in resistance to treatments, recurrence and distant metastasis and eradication of CSCs/CICs is crucial to improve recent therapy. Cytotoxic T lymphocytes (CTLs) are major effectors of cancer immunotherapy, and CTLs recognize antigenic peptides derived from antigens that are presented by major histocompatibility complex (MHC) class I molecules. In this study, we analyzed the potency of a cancer-testis (CT) antigen, brother of the regulator of the imprinted site variant subfamily 6 (BORIS sf6), in lung CSC/CIC immunotherapy. BORIS sf6 mRNA was expressed in lung carcinoma cells (9/19), especially in sphere-cultured lung cancer stem-like cells, and in primary lung carcinoma tissues (4/9) by RT-PCR. Immunohistochemical staining using BORIS sf6-specific antibody revealed that high expression of BORIS sf6 is related to poorer prognosis. CTLs could be induced by using a human leukocyte antigen, (HLA)-A2 restricted antigenic peptide (BORIS C34_24(9)), from all of 3 HLA-A2-positive individuals, and CTL clone cells specific for BORIS C34_24(9) peptide could recognize BORIS sf6-positive, HLA-A2-positive lung carcinoma cells. These results indicate that BORIS sf6 is a novel target of lung cancer immunotherapy that might be useful for targeting treatment-resistant lung cancer stem-like cells

Induction of BORIS subfamily 6-specific CTLs and establishment of a CTL clone.

<p>BORIS subfamily 6 peptide-specific cytotoxic T cell (CTL) induction was evaluated by the interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay. HLA-A*0201-positive PBMCs were obtained from four healthy donors. Donors A, B and C were HLA-*A0201-positive. Fluorescence-activated cell sorting (FACS) was performed with PE-conjugated BORIS subfamily 6 peptide/HLA-A*0201 tetramer and anti-CD8-FITC antibody. Double positive cells were single-cell sorted to establish a CTL clone (total 960 well). Twelve wells showed cell growth and 8 wells were analyzed by tetramer for specificity.</p

BORIS subfamily 6 expression in lung cancer cell lines.

<p><b>(A) An image of sphere-cultured SBC5 cells.</b> Magnification, x 400. Bar scale is 100 μm. <b>(B) Expression levels of stem cell-related genes.</b> Expression levels of stem cell-related genes including ABCG2, ALDH1A1, NANOG, POU5F1, SOX2, KLF4 and BMI1 were determined by quantitative RT-PCR. Data are shown as means ± SD. All statistical analyses for data shown in this figure were performed using bilateral Student’s t test. *P-values <0.05. <b>(C) Expression of SOX2 protein in sphere-cultured cells.</b> SOX2 protein expression in sphere-cultured and adherent-cultured SBC5 cells were analyzed by an Western blot. β-Actin was used as a positive control. <b>(D) Expression of the cancer testis antigen BORIS sf6 in adherent-cultured and sphere-cultured lung carcinoma cells.</b> RT-PCR analysis of BORIS subfamily 6 mRNA expression in lung cancer cell lines and primary cancer cells from clinical specimens. Cancer cells were cultured in adherent culture and sphere culture.</p

BORIS subfamily 6-specific CTL clone analysis.

<p><b>(A) ELISPOT assay of BORIS subfamily 6-specific CTL clones 3B9 and 4D2 clones. (B) Tetramer assay of BORIS subfamily 6-specific CLT clones 3B9 and 4D2.</b> CTL clones 3B9 and 4D2 were stained by PE-conjugated BORIS subfamily 6-specific peptide/HLA-A*0201 tetramer and anti-CD8-FITC antibody and analyzed. <b>(C) LDH release cytotoxicity assay.</b> Specific cytotoxicity for peptide-pulsed T2 cells was displayed (left panel). HIV peptide-pulsed T2 cells, peptide (-) T2 cells and K562 cells were used as negative controls. Specific cytotoxicity for HLA-A2-positive lung cancer cell lines SBC5 and LC142 was displayed (right panel). Data are shown as means ± SD. All statistical analyses for data shown in this figure were performed using bilateral Student’s t test. *P-values <0.05.</p

Expression of BORIS sf6 and characteristics of lung cancer patients.

<p>Expression of BORIS sf6 and characteristics of lung cancer patients.</p