Except for melanomas, tumor antigens recognized by cytotoxic T lymphocytes (CTLs) are yet unidentified. We have identified a gene encoding antigenic peptides of human squamous cell carcinomas (SCCs) recognized by human histocompatibility leukocyte antigens (HLA)- A2601–restricted CTLs. This gene showed no similarity to known sequences, and encoded two (125- and 43-kilodalton [kD]) proteins. The 125-kD protein with the leucine zipper motif was expressed in the nucleus of the majority of proliferating cells tested, including normal and malignant cells. The 43-kD protein was expressed in the cytosol of most SCCs from various organs and half of lung adenocarcinomas, but was not expressed in other cancers nor in a panel of normal tissues. The three nonapeptides shared by the two proteins were recognized by the KE4 CTLs, and one of the peptides induced in vitro from peripheral blood mononuclear cells (PBMCs) the CTLs restricted to the autologous tumor cells. The 43-kD protein and this nonapeptide (KGSGKMKTE) may be useful for the specific immunotherapy of HLA-A2601+ epithelial cancer patients.

The epithelial cell transforming gene 2 (ECT2) protooncogene encodes a Rho exchange factor, and regulates cytokinesis. ECT2 is phosphorylated in G2/M phases, but its role in the biological function is not known. Here we show that two mitotic kinases, Cdk1 and polo-like kinase 1 (Plk1), phosphorylate ECT2 in vitro. We identified an in vitro Cdk1 phosphorylation site (T412) in ECT2, which comprises a consensus phosphospecific-binding module for the Plk1 polo-box domain (PBD). Endogenous ECT2 in mitotic cells strongly associated with Plk1 PBD, and this binding was inhibited by phosphatase treatment. A phosphorylation-deficient mutant form of ECT2, T412A, did not exhibit strong association with Plk1 PBD compared with wild-type (WT) ECT2. Moreover, ECT2 T412A, but not phosphomimic T412D, displayed a diminished accumulation of GTP-bound RhoA compared with WT ECT2, suggesting that phosphorylation of Thr-412 is critical for the catalytic activity of ECT2. Moreover, while overexpression of WT ECT2 or the T412D mutant caused cortical hyperactivity in U2OS cells during cell division, this activity was not observed in cells expressing ECT2 T412A. These results suggest that ECT2 is regulated by Cdk1 and Plk1 in concert.

Cleavage furrow formation marks the onset of cell division during early anaphase. The small GTPase RhoA and its regulators ECT2 and MgcRacGAP have been implicated in furrow ingression in mammalian cells, but the signaling upstream of these molecules remains unclear. We now show that the inhibition of cyclin-dependent kinase (Cdk)1 is sufficient to initiate cytokinesis. When mitotically synchronized cells were treated with the Cdk-specific inhibitor BMI-1026, the initiation of cytokinesis was induced precociously before chromosomal separation. Cytokinesis was also induced by the Cdk1-specific inhibitor purvalanol A but not by Cdk2/Cdk5-or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of antiCdk1 monoclonal antibody resulted in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibited BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induced excessive membrane blebbing, which appeared to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolished both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduced both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs. We propose that Cdk1 inactivation is sufficient to activate a signaling pathway leading to cytokinesis, which emanates from mitotic spindles and is regulated by ECT2, MgcRacGAP, and RhoA. Chemical induction of cytokinesis will be a valuable tool to study the initiation mechanism of cytokinesis.

Polymorphisms in the 5'-flanking promoter/enhancer region of the tumor necrosis factor (TNF)-a gene were examined to study the genetic background of rheumatoid arthritis (RA). Four variant alleles, -1,031C/ -863A, -1,031C/-238A, -857T and -308A, were identified and examined in 387 RA patients and 575 healthy Japanese controls. The frequency of the -857T allele in RA patients was significantly higher than that in the controls. However, the HLA-DRB1 analysis in the same subjects showed that the DRB1*0405 allele, which is in linkage disequilibrium with the -857T, was more strongly associated with the disease susceptibility than the -857T allele. These results suggest that the susceptible gene to RA is more closely linked to the HLA-DRB1 locus than to the TNF-alpha locus.

Development of therapeutic vaccines is one of the major areas of tumour immunotherapy today. However, clinical trials of peptide-based cancer vaccines have rarely resulted in tumour regression. This failure might be due to an insufficient induction of cytotoxic T lymphocytes in the current regimes, in which cytotoxic T lymphocytes-precursors in pre-vaccination peripheral blood mononuclear cells are not measured. Initiation of immune-boosting through vaccination could be better than that of immune-priming with regard to induction of prompt and strong immunity. If this is also the case for therapeutic vaccines, prevaccination measurement of peptide-specific cytotoxic T lymphocytes-precursors will be important. In the present study, we investigated whether cytotoxic T lymphocytes-precursors reacting to 28 kinds of peptides of vaccine candidates (13 and 15 peptides for HLA-A24 + and HLA-A2 + patients, respectively) were detectable in pre-vaccination peripheral blood mononuclear cells of 80 cancer patients. Peptide-specific cytotoxic T lymphocytes-precursors were found to be detectable in peripheral blood mononuclear cells of the majority of cancer patients (57 out of 80 cases, 71%). The mean numbers of positive peptides were 2.0 peptides per positive case. Peripheral blood mononuclear cells incubated with positive peptides, not with negative peptides, showed significant levels of HLA-class-I-restricted cytotoxicity to cancer cells. The profiles of positive peptides entirely varied among patients, and were not influenced by the cancer origin. These results may provide a scientific basis for the development of a new approach to cancer immunotherapy, e.g.) cytotoxic T lymphocytes-precursororiented peptide vaccine.