Toshiyoshi Fujiwara

Wild-Type Human p53 and a Temperature-Sensitive Mutant Induce Fas/APO-1 Expression

Fas/APO-1 is a cell surface protein known to trigger apoptosis upon specific antibody engagement. Because wild-type p53 can activate transcription as well as induce apoptosis, we queried whether p53 might upregulate Fas/APO-1. To explore this possibility, we examined human p53-null (H358 non-small-cell lung adenocarcinoma and K562 erythroleukemia) and wild-type p53-containing (H460 non-small-cell lung adenocarcinoma) cell lines. When H358 or H460 cells were transduced with a replication-deficient adenovirus expression construct containing the human wild-type p53 gene but not with vector alone, a marked upregulation (approximately a three-to fourfold increase) of cell surface Fas/APO-1 was observed by flow cytometry. Similarly, K562, cells stably transfected with a plasmid vector containing the temperature-sensitive human p53 mutant Ala-143 demonstrated a four- to sixfold upregulation of Fas/APO-1 by flow-cytometric analysis at the permissive temperature of 32.5 degrees C. Temperature-sensitive upregulation of Fas/APO-1 in K562 Ala-143 cells was verified by immunoprecipitation and demonstrated to result from enhanced mRNA production by nuclear run-on and Northern (RNA) analyses. Stably transfected K562 cells expressing temperature-insensitive, transcriptionally inactive p53 mutants (His-175, Trp-248, His-273, or Gly-281) failed to upregulate Fas/APO-1 at either 32.5 degrees or 37.5 degrees C. The temperature-sensitive transcription of Fas/APO-1 occurred in the presence of cycloheximide, indicating that de novo protein synthesis was not required and suggested a direct involvement of p53. Collectively, these observations argue that Fas/APO-1 is a target gene for transcriptional activation by p53.

Gene therapeutics and gene therapy for cancer.

Recent advances in molecular biology have opened new avenues of basic genetic engineering technology and have made possible the application of this technology in clinical human gene therapy. Replication-defective viral vectors and biocompatible materials, eg, liposomes, have been developed as vehicles to introduce potentially therapeutic genes into mammalian cells. Over the past 2 years, this technology has increased the possibilities for therapy in numerous genetic diseases. Approaches at the molecular level have also demonstrated that one of the mechanisms of human cancer development is overexpression of dominant oncogenes, expression of mutant oncogenes, or specific chromosomal deletions or mutations that induce inactivation of tumor-suppressor activity. This concept suggests that the introduction of antisense oncogenes and wild-type tumor-suppressor genes, eg, p53, could halt or reverse these mechanisms, thus having a therapeutic effect in cancer. Moreover, evidence that the immune system is capable of eliminating tumor cells in numerous animal models has suggested gene therapy approaches for the delivery of cytokines, which promote the activation of cytotoxic immune responses against the malignant tissue. The efficacy of these gene therapy protocols is now being evaluated in both animal model systems and clinical trials. This article reviews recent highlights in this rapidly evolving field.

Advances in Cancer Gene Therapy

Publisher Summary The potential effectiveness of gene therapy in cancer treatment is promised not only by its precise targeting of the mechanisms of the disease, but also by its genetic approaches that are based on rapidly advancing molecular biotechnology. Diverse strategies and innovative approaches to cancer gene therapy have been developed. Despite the great potential and rapid advances in this technology, the development of cancer gene therapy is at a very early stage. This chapter presents an overview of the current state of this field, for understanding the prospects of cancer gene therapy. Nonviral gene delivery systems are one of the current approaches to gene transfer. There is a variety of effective nonviral techniques for transfection of DNA into mammalian cells in vitro, such as calcium-phosphate precipitation, polycation-mediated transfection, protoplast fusion, electroporation, microinjection, and liposome- or ligand-mediated gene transfer. The last two methods have the greatest potential to be further developed for the purpose of in vivo gene transfer. Moreover, direct injection of naked DNA into susceptible tissues is a unique approach for nonviral in vivo gene transfer. Other than nonviral approach there are also other approaches such as recombinant virus-mediated gene transfer. For the purpose of cancer gene therapy, ideal viral vectors are considered that have the ability to target gene delivery, have a large gene-carrying capacity, allow highly efficient gene transfer, allow controllable or tissue-specific gene expression, have high therapeutic potentials, and have low immunogenicity and cytotoxicity. The explosive advances in research of gene regulation of cells and molecular mechanisms of diseases have aided in the design of drugs that target genetic sequences through specific recognition of and by hydrogen bonding among complementary bases. Along the pathway for genetic information, flow from DNA to protein, several approaches have been developed to modulate gene expression and regulation.

Gene Replacement Strategies for Lung Cancer.

Advances in our understanding of the molecular genetics of cancer present an opportunity to develop prevention and treatment strategies based on the reversal of specific genetic lesions. This strategy is analogous to the classic concept of gene therapy for replacement of defective or nonfunctioning genes. The gene families implicated in carcinogenesis include dominant oncogenes and tumor suppressor genes. Regional administration of viral vectors expressing wildtype p53 and antisense K-ras prevents tumor growth for tumors with the specific genetic lesions in orthotopic tumor models and mediates regression of large established tumors. These studies provide a rationale for a new clinical protocol recently approved by the National Institutes of Health Recombinant DNA Advisory Committee and Food and Drug Administration to replace a defective p53 gene with intratumor injection of recombinant retrovirus expressing wild-type p53 or elimination of activated K-ras by expression of antisense K-ras messenger RNA. If these agents are efficacious, their lack of toxicity may provide a sufficiently high therapeutic index such that they could be used as an adjuvant to surgery to treat patients with earlier stages of cancer or as prevention for second primary cancers for individuals with genetic abnormalities in premalignant lesions. Although much research needs to be done, the possibility of specific gene targeting with a high therapeutic index makes this a promising area of investigation.