Cellular tumor antigen p53 (217-225)

Tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2) has been considered as a rational target for gene therapy in solid tumors. In order to identify a novel peptide ligand of Tie2 for targeted gene therapy, we screened a phage display peptide library and identified a candidate peptide ligand NSLSNASEFRAPY (designated GA5). Binding assays and Scatchard analysis revealed that GA5 could specifically bind to Tie2 with a dissociation constant of 2.1x10(-8)M. In addition, we showed that GA5 was internalized into tumor cells highly expressing Tie2. In the biodistribution assay, (125)I-GA5 was mainly accumulated in SPC-A1 xenograft tumors that express Tie2. In gene delivery studies, GA5-conjugated polyethylenimine vector could achieve greater transgene transduction than non-targeted vectors both in vitro and in vivo. Tumor growth inhibition was observed in SPC-A1 xenograft-bearing mice that received eight intratumoral injections of GA5-polyethylenimine/p53 complexes in 3 weeks. The difference in tumor volume between the experiment and control groups was significant (P<0.05). Our results showed that GA5 is a potentially efficient targeting element for cancer gene or molecular therapy.

In spite of the benign nature of subependymal giant cell astrocytomas (SEGAs), some show massive hemorrhage, rapid growth, and tumor recurrence. This led us to investigate the biological behavior, cell dynamics, and tumorigenesis of SEGAs. All patients (4 men and 3 women; age range, 6-27 years; mean, 13.6 years) had features of tuberous sclerosis complex and obstructive hydrocephalus. One patient had intratumoral bleeding. In two patients, sequential neuroimaging showed a subependymal nodule growing to become a SEGA. All underwent surgical resection without radiation therapy. One tumor recurred and was treated surgically. There were no postoperative deaths. The presence of cytologic atypia, mitoses and vascular proliferation had no implication in terms of the clinical course. MIB-1 labeling indices were low (mean, 0.9), indicating low proliferative potential. Unexpectedly, bcl-2 staining was sparse and bax staining predominated in majority of cases. However, the mean value of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling index was low. Immunohistochemically, tumors were positive for both glial and neuronal markers. In the majority of our cases, the expression of p53 was low. Only one tumor was focally positive for tuberin. SEGAs have low proliferative potential and apoptotic activity, and exhibit features of mixed glial-neuronal differentiation. In contrast to p53, tuberin is suggested to be the tumor suppressor in this tumor.

The tumour-suppressor gene p53 is pivotal in the regulation of apoptosis, and point mutations within p53 are the commonest genetic alterations in human cancers. Cytotoxic T lymphocytes (CTL) recognise peptide-MHC complexes on the surface of tumour cells and bring about lysis. Therefore, p53-derived peptides are potential candidates for immunisation strategies designed to induce antitumour CTL in patients. Conformational changes in the p53 protein, generated as a result of point mutations, frequently expose the 240 epitope, RHSVV (amino acids 212-217), which may be processed differently from the wild-type protein resulting in an altered MHC-associated peptide repertoire recognised by tumour-specific CTL. In this study 42 peptides (37 overlapping nonameric peptides, from amino acids 193-237 and peptides 186-194, 187-197, 188-197, 263-272, 264-272, possessing binding motifs for HLA-A2) derived from the wild-type p53 protein sequence were assayed for their ability to stabilise HLA-A2 molecules in MHC class I stabilisation assays. Of the peptides tested, 24 stabilised HLA-A2 molecules with high affinity (fluorescence ratio >1.5) at 26 degrees C, and five (187-197, 193-200, 217-224, 263-272 and 264-272) also stabilised the complexes at 37 degrees C. Peptides 188-197, 196-203 and 217-225 have not previously been identified as binders of HLA-A2 molecules and, of these, peptide 217-225 stabilised HLA-A2 molecules with the highest fluorescence ratio. Peptide 217-225 was chosen to generate HLA-A2-restricted CTL in vitro; peptide 264-272 was used as a positive control. The two primary CTL thus generated (CTL-217 using peptide 217 225; and CTL-264 using peptide 264-272) were capable of specifically killing peptide-pulsed T2 or JY cells. In order to determine whether these peptides were endogenously processed and to test the hypothesis that mutants expressing different protein conformations would generate an alternative peptide repertoire at the cell surface, a panel of target cells was generated. HLA-A2+ SaOs-2 cells were transfected with p53 cDNA containing point mutations at either position 175 (R-->H) or 273 (R-->H) (SaOs-2/175 and SaOs-2/273). Two HLA-A2-negative cell lines, A431 and SKBr3, naturally expressing p53 mutations at positions 273 and 175 respectively, were transfected with a cDNA encoding HLA-A2. The results showed that primary CTL generated in response to both peptides were capable of killing SaOs-2/175 and SKBr3-A2 cells, which possess the same mutation, but not SaOs-2/273, A431-A2 or SKBr3 cells transfected with control vector. This suggests that these peptides are presented on the surface of SaOs-2/175 and SKBr3-A2 cells in a conformation-dependent manner and represent potentially useful target peptides for immunotherapy.