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The eEF1〈-2 gene (S1) encodes a tissue-specific isoform of peptide elongation factor-1A (eEF1A-1); its mRNA is expressed only in brain, heart, and skeletal muscle, tissues dominated by terminally differentiated, long-lived cells. Homozygous mutant mice exhibit muscle wasting and neurodegeneration, resulting in death around postnatal day 28. eEF1〈-2/S1 protein shares 92% identity with eEF1A-1; because specific antibodies for each were not available previously, it was difficult to study the developmental expression patterns of these two peptide elongation factors 1A in wasted and wildtype mice. We generated a peptide-derived antiserum that recognizes the eEF1〈-2/S1 isoform and does not cross-react with eEF1A-1. We characterized the expression profiles of eEF1A-1 and eEF1A-2/S1 during development in wild-type (؉/؉), heterozygous (؉/wst), and homozygous (wst/wst) mice. In wild-type and heterozygous animals, eEF1A-2/S1 protein is present only in brain, heart, and muscle; the onset of its expression coincides with a concomitant decrease in the eEF1A-1 protein level. In wasted mutant tissues, even though eEF1A-2/S1 protein is absent, the scheduled decline of eEF1A-1 occurs nonetheless during postnatal development, as it does in wild-type counterparts. In the brain of adult wild-type mice, the eEF1A-2/S1 isoform is localized in neurons, whereas eEF1A-1 is found in non-neuronal cells. In neurons prior to postnatal day 7, eEF1A-1 is the major isoform, but it is later replaced by eEF1A-2/S1, which by postnatal day 14 is the only isoform present. The postdevelopmental appearance of eEF1A-2/S1 protein and the decline in eEF1A-1 expression in brain, heart, and muscle suggest that eEF1A-2/S1 is the adult form of peptide elongation factor, whereas its sister is the embryonic isoform, in these tissues. The absence of eEF1A-2/S1, as well as the on-schedule development-dependent disappearance of its sister gene, eEF1A, in wst/ wst mice may result in loss of protein synthesis ability, which may account for the numerous defects and ultimate fatality seen in these mice.Peptide elongation factor-1A (eEF1A-1) 1 is an abundant protein, once thought to be ubiquitously expressed; its major role is to mediate the transfer of charged aminoacyl-tRNA to the A site of the ribosome during peptide elongation. We have previously shown that the mRNA expression of this gene declines in rat brain, heart, and skeletal muscle during development (1, 2), to the extent that in adult skeletal muscle, there is an almost complete loss of eEF1A-1 mRNA expression; a sister gene, eEF1A-2/S1, is expressed instead (3). Furthermore, in contrast to the ubiquitous expression of eEF1A-1 in many cell types, eEF1A-2/S1 expression is limited to the terminally differentiated cells of the brain, heart, and skeletal muscle (3, 4).Recent evidence suggests that eEF1A-2/S1 is an alternative peptide elongation factor-1A for neurons and myocytes, because the amino acid homology between them is 92% (5, 6), and eEF1A-2/S1 can perform peptide elongation in vitro (7). The absence ...

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Our study has shown that p53 status is important for CD::UPRT/5-FC treatment. Moreover, this study demonstrated that the three-dimensional spheroid model is a more stringent in vitro model for suicide gene therapy evaluation than are monolayer cultures.

A promising approach for cancer gene therapy is the combination of adenovirus vectors (AdV) with the suicide gene cytosine deaminase and uracil phosphoribosyl transferase (CDHUPRT). While such vectors have been tested in tumor cell lines and xenograft models, it is not clear how these therapeutic vectors would perform in primary human tumors. We, thus, examined the effect of the combination of a recombinant adenovirus expressing the CDHUPRT (AdCU) with 5-fluorocytosine (5-FC) on primary cancer cells isolated from the ascites or pleural fluids of patients with metastatic cancers. In such models, we have found a direct correlation between the patients' response to 5-FU and the response shown by the cancer cells in vitro, confirming the clinical relevance of this methodology. Our findings demonstrated that this combination was able to kill primary tumor cells, including those that had developed resistance to 5-FU. Furthermore, while proliferating cells were more susceptible to 5-FU, the combination was effective in both rapid and slow proliferating samples. Our study demonstrated that this gene therapy approach could provide an effective therapeutic option for cancers and is not affected by acquired 5-FU resistance. Also of importance is the effectiveness of this gene therapy approach on slower proliferating cells that is typical of the majority of cancers in vivo. This suggests a greater likelihood that it will be effective in a clinical setting.

Gene therapy is a promising approach for cancer treatment; however, efficacy of current vectors remains insufficient. To improve the success of suicide gene therapy, we constructed a replication-competent adenoviral vector that has its protease gene deleted and expresses bacterial cytosine deaminase fused with bacterial uracil phosphoribosyltransferase (CU). The prodrug, 5-fluorocytosine, is transformed into the highly toxic and tissue-diffusible 5-fluorouracil by CU in infected cells. This vector is incapable of producing infectious particles but is able to undergo a single round of replication, thereby increasing transgene copy number and expression. In the presence of 5-FC, compared with the first-generation vector (AdCU), the replication-competent vector, Ad(dPS)CU-IRES-E1A, was significantly more efficacious for in vitro tumor cell killing and in bystander assays, whereas 25-fold fewer viral particles were required in a three-dimensional spheroid model. For in vivo experiments, in which virus was injected into preestablished intracranial glioma xenografts, followed by 5-FC treatment, mice receiving Ad(dPS)CU-IRES-E1A had significantly smaller tumors at 35 days postinjection as well as significantly longer median survival than mice treated with the replication-deficient, protease-deleted vector [Ad(dPS)CU]. In an immunocompetent syngeneic model, Ad(dPS)CU + 5-FC-treated mice had a median survival of only 23 days, whereas Ad(dPS)CU-IRES-E1A + 5-FCtreated animals had a survival of 57.1% at 365 days. In conclusion, Ad(dPS)CU-IRES-E1A in the presence of 5-FC produces more potent tumoricidal effects than its replicationdeficient counterparts. [Cancer Res 2007;67(7):3387-95]