A major problem facing the effective treatment of patients with cancer is how to get the specific antitumor agent into every tumor cell. In this report we describe the use of a strategy that, by using retroviral vectors encoding a truncated human CD5 cDNA, allows the selection of only the infected cells, and we show the ability to obtain, before bone marrow transplantation, a population of 5-f luouraci-treated murine bone marrow cells that are 100% marked. This marked population of bone marrow cells is able to reconstitute the hematopoietic system in lethally irradiated mice, indicating that the surface marker lacks deleterious effects on the functionality of bone marrow cells. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD5-expressing cells. Nevertheless, a significant proportion of the hematopoietic cells no longer expresses the surface marker CD5 in the 9-month-old recipient mice. This transcriptional inactivity of the proviral long terminal repeat (LTR) was accompanied by de novo methylation of the proviral sequences. Our results show that the use of the CD5 as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, which can entirely reconstitute the hematopoietic system in mice. These results should now greatly enhance the power of studies aimed at addressing questions such as generation of cancer-negative hematopoiesis.Recombinant retroviruses provide an attractive vehicle for gene transfer based on their capacity for highly efficient infection and nontoxic integration into the genome of a wide range of cell types. Although the potential for retrovirusmediated gene transfer efficiency approaches 100%, this may not be realized because of low viral titers or the failure to stimulate cells to divide, which is required for successful integration. Moreover, expression of the transferred gene(s) may not reach desired levels or be sustained. Such problems are evident in current efforts to apply retroviral gene transfer to the gene therapy of cancer, because if we cannot deliver the antitumor specific drug (1-3) into every tumor cell, then any malignant cell that remains unaffected will emerge as a resistant clone. Recently we have tested the utility of coexpressing the specific antitumor agent and a cDNA encoding a truncated human cell surface antigen CD5 for the immediate postinfection selection of the hematopoietic cells transduced with the retrovirus containing the specific antitumor drug (4). Fluorescence-activated cell sorter (FACS) analysis in combination with functional studies showed that under the conditions used, all cells selected expressed CD5 within 48 hr of termination of the infection procedure. Moreover, the fraction of CD5-positive cells did not decrease with extended propagation of selected cells, indicating that the surface marker lacks deleterious effects on cell growth effect and viability. The use of the truncated CD5 cell surface antigen as a selectable marker of gene transfer offers s...
Background: The main difficulty in providing effective treatment of patients with cancer is distinguishing between tumor and normal cells. The chimeric molecules created by cancer-associated chromosomal abnormalities (such as the BCR-ABL fusion protein) represent ideal therapeutic targets since they are unique to the disease state. A major challenge, however, is how to deliver the specific anti-tumor agent into every tumor cell.Material and Methods: In this report we describe the use of a novel strategy to introduce specific anti-tumor reagents into every tumor cell. It uses retroviral vectors encoding both antisense transcripts specific for the BCR-ABLP'90 fusion junction (the specific anti-tumor drug) and a truncated human CD5 cDNA, which allows selection of the infected cells. In order to coexpress the anti-sense molecule with the truncated human CD5 gene, the picomavirus internal ribosome-entry site was incorporated in the constructs. Results: When the antisense transcripts in the CD5retroviral vector were introduced into Ba/F3+pl90 cells rendered interleukin 3 (IL-3) independent by expression of the BCR-ABL sequences, the cells died upon IL-3 withdrawal, as measured by the absence of CD5-positive cells. Control Ba/F3+p210 cells infected with the same virus did not die in the absence of IL-3. Conclusions: These data suggest a novel strategy for cancer treatment which incorporates the use of a retrovirus coexpressing both a selectable surface marker and a tumor-specific agent.
TXNIP is a protein sensitive to oxidant conditions whose expression is related to the progression of death in cancer, diabetes, ischemia, and neurodegenerative diseases, among others. Because of this, many studies propose TXNIP as a therapeutic target in several diseases. Exposure of cerebellar granule neurons to staurosporine or low potassium leads to apoptotic death. Both conditions generate an early production of reactive oxygen species (ROS) that induces the activation of the ASK1 pathway and the apoptotic machinery. In these models, it has been shown an increase in TXNIP protein mediated by ROS. Here, we evaluated the molecular mechanisms involved in the regulation of the Txnip expression during neuronal death, as well as the role of the protein in the progression of cell death induced by these two apoptotic conditions. In cultured cerebellar granule neurons, we observed that low potassium and staurosporine induced an early increase in ROS that correlated with an increase in Txnip mRNA. When we evaluated the promoter of the gene, we found that the JASPAR-reported FOXO1/3 transcription factor motifs are close to the transcription start site (TSS). We then verified through the Chromatin immunoprecipitation technique (ChIP) that FOXO3 interacts with the Txnip promoter after 1 h of low potassium treatment. We also detected FOXO3 nuclear translocation by low potassium and staurosporine treatments. Finally, by using shRNA in the neuroblastoma MSN cell line, we found that Txnip downregulation decreased neuronal death induced by staurosporine stimulus. Together, these results suggest that ROS promotes the expression of Txnip through the activation of the FOXO3 transcription factor mediated by Akt inhibition. We also demonstrated that TXNIP is necessary for neuronal death progression.
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