Encoding Tumor Antigens Research Articles

Induction of MAGE Genes in Lymphoid Cells by the Demethylating Agent 5‐Aza‐2′‐deoxycytidine

MAGE genes encoding tumor antigens recognized by cytotoxic T lymphocytes are appropriate target molecules for specific immunotherapy of cancer. We have investigated whether the demethylating agent 5‐aza‐2′‐deoxycytidine (DAC) induces MAGE‐1, ‐2, ‐3, and ‐6 in normal and malignant lymphoid cells. DAC induced these MAGE genes in both PHA/interleukin‐2 (IL‐2)‐activated T cells from healthy donors and MAGE‐negative T and B cell leukemias in most cases. It also induced MAGE‐1 in IL‐2‐dependent T cell clones and all MAGE genes tested in Epstein‐Barr virus‐transformed B cell lines. Expression of MAGE‐1 protein in the cells was confirmed by western blot analysis with anti‐MAGE‐1 polyclonal antibody. Therefore, demethylation is a potent stimulus to induce MAGE genes in both normal and malignant lymphoid cells.

Generation and synchronization of gonadotropin-releasing hormone (GnRH) pulses: intrinsic properties of the GT1-1 GnRH neuronal cell line.

The immortalized neuronal cell line GT1-1 was used to investigate the endogenous pattern of GnRH release. The GT1-1 cell line was derived from a GnRH-secreting tumor in a transgenic mouse induced by genetically targeted expression of the potent simian virus 40 oncogene encoding tumor antigen. Cells attached to coverslips were superfused in Sykes-Moore chambers with Locke's medium, Ca(2+)-free Locke's medium, or Opti-MEM (another defined medium) for 2 hr, and samples were collected at 4-min intervals. Release of GnRH in 17 of 18 superfusion chambers was seen to be pulsatile when data were analyzed by cluster analysis. No significant differences were observed whether only one or both of the coverslips forming the chamber were coated with cells. Pulses exhibited a mean interpulse interval of 25.8 +/- 1.5 min, a mean duration of 18.8 +/- 1.4 min, and a mean amplitude of 150.5 +/- 6.0% above preceding nadir. The removal of Ca2+ from the Locke's medium resulted in the progressive reduction of the amplitude and eventually in the absence of identifiable pulses. Pulses reappeared after the return of Ca2+ to the medium. It is concluded that the GT1-1 cell line secretes GnRH in a rhythmic pattern. These findings suggest that the pulsatile release of GnRH (GnRH pulse generator) may be an intrinsic characteristic of the GnRH neurons. Synchronization of pulsatile release from individual neurons could be mediated via numerous cell-to-cell contacts observed in the cultured cells on coverslips. Synchronization of GnRH release from cells on two physically separated coverslips forming a chamber would appear to be accomplished by a diffusible mediator.

The role of the immune system in the pathogenesis of cancer

The observation that malignant cells express antigens that may be recognized by immunocytes and that immune effector mechanisms have the capability of destroying tumor cells has increased our appreciation of the biology of cancer and its relationship to immune function as well as offered new options for therapeutic intervention. Clinical trials are in progress to evaluate several different approaches to modifying the host's immune response against tumor. One approach is to administer agents that have direct activity against the malignancy. For example, antibody conjugates bring cytotoxic molecules of chemotherapy, radioisotopes, or toxins directly to the tumor. 4 4 Abbas AK, Lichtman AH, Pober JS (eds): Cellular and Molecular Immunology. Philadelphia, PA, Saunders, 1991. A second approach is to administer agents that modulate the host's own antitumor response such as IFN-α and IFN-gg. 4 Adoptive cellular immunotherapy aimed at isolating and expanding the host's own tumor-specific lymphocytes and inducing activation and proliferation with lymphokines such as IL-2 has shown encouraging results. 1 1 Greenberg PD: Mechanisms of tumor immunology, in Stites DP, Terr AI (ed): Basic and Clinical Immunology. Norwalk, CT, Appleton & Lange, 1991, pp 580–587. Even though clinical data are still quite premature, it is reasonable to assume that in the future immunomodulation including the stimulation of immune effector mechanisms to eradicate tumor, the reconstitution of immune deficiency in diseases such as AIDS, the suppression of immune function to avoid graft rejection and GVHD, 41 41 Jaffe HS, Sherwin SA: Immunomodulators, in Stites DP, Terr AI (eds): Basic and Clinical Immunology. Norwalk, CT, Appleton & Lange, 1991, pp 73–77. and the isolation and insertion of genes encoding tumor antigens into recombinant vectors to immunize the host to the tumor antigen will be commonly and successfully employed. 1

Inactivation of p53 gene expression by an insertion of Moloney murine leukemia virus-like DNA sequences.

Analysis of Abelson murine leukemia virus-transformed L12 cells which lack the p53 cellular encoded tumor antigen revealed alterations in the p53-specific genomic DNA sequences. The active p53 gene, usually contained in a 16-kilobase EcoRI DNA fragment of p53 producer cells, went through major alterations leading to the appearance of a substantially larger 28.0-kilobase p53-specific EcoRI fragment. Detailed restriction enzyme analysis, with genomic probes spanning throughout the whole active p53 gene, indicated that the L12 p53 altered gene contains all the exons and principal introns of the normal p53 16.0-kilobase gene. However, its structure was interrupted by the integration of a novel DNA segment into the noncoding intervening sequences of the first p53 intron. Analysis of the inserted sequences revealed close homology to Moloney murine leukemia virus. This Moloney leukemia murine virus-like particle resides in a 5' to 3' transcriptional orientation, similar to the p53 gene, permitting the transcription of aberrant fused mRNA molecules detected in these cells.

Inactivation of p53 gene expression by an insertion of Moloney murine leukemia virus-like DNA sequences.

Analysis of Abelson murine leukemia virus-transformed L12 cells which lack the p53 cellular encoded tumor antigen revealed alterations in the p53-specific genomic DNA sequences. The active p53 gene, usually contained in a 16-kilobase EcoRI DNA fragment of p53 producer cells, went through major alterations leading to the appearance of a substantially larger 28.0-kilobase p53-specific EcoRI fragment. Detailed restriction enzyme analysis, with genomic probes spanning throughout the whole active p53 gene, indicated that the L12 p53 altered gene contains all the exons and principal introns of the normal p53 16.0-kilobase gene. However, its structure was interrupted by the integration of a novel DNA segment into the noncoding intervening sequences of the first p53 intron. Analysis of the inserted sequences revealed close homology to Moloney murine leukemia virus. This Moloney leukemia murine virus-like particle resides in a 5' to 3' transcriptional orientation, similar to the p53 gene, permitting the transcription of aberrant fused mRNA molecules detected in these cells.