In vitro evidence for a new therapeutic approach in renal cell carcinoma

PURPOSE: Renal cell carcinoma (RCC) is the most lethal among the common urologic malignancies, comprising 3% of all human neoplasias; approximately 40% of patients eventually die of cancer progression. One third of patients who present with metastatic disease and up to 40% treated for localized disease generally experience recurrence. RCCs are characterized by high resistance to chemo-, radio- and immunotherapy. We recently discovered an endogenous enzymatic activity, which is particularly expressed in tumorigenic cell, endogenous non-telomerase reverse transcriptase (RT) of retrotrasposon / retroviral origin, as a specific target to induce proliferation arrest in a number of human carcinogenesis in vitro culture cell lines. METHODS: To address this possibility, we have employed RCC primary cell culture testing pharmacological inhibition, in vitro, by two characterized non nucleosidic RT inhibitors, nevirapine and efavirenz; next, we assessed morphological effects and analyzed putative modulation on gene expression profile. RESULTS: Both treatments reduced cell proliferation rate and induced morphological differentiation and gene expression reprogramming in different RCC analyzed tumor biomarkers. CONCLUSION: In this study we describe a new potential therapeutic approach to obtain considerable future benefits in renal carcinoma cure and attempt to establish a new possible pharmacological therapy based on oral drugs administration in renal RCC treatment

Generation of biologically active retro-genes upon interaction of mouse spermatozoa with exogenous DNA

Mature spermatozoa of most animal species can spontaneously take up foreign DNA molecules which can be delivered to embryos upon fertilization. Following this procedure, transgenic animals of various species have been generated. We recently discovered a reverse transcriptase (RT) activity in mouse spermatozoa that can reverse-transcribe exogenous RNA molecules into cDNA copies. These cDNA copies are transferred to embryos at fertilization, mosaic propagated as non-integrated structures in tissues of founder individuals and further transmitted to F1 progeny. Reverse-transcribed sequences behave as functional genes, being correctly expressed in tissues of F0 and F1 animals. To learn more about this mechanism and further characterize the reverse transcription step, we have now incubated spermatozoa with a plasmid harboring a green fluorescent protein (EGFP) retrotransposition cassette interrupted by an intron in the opposite orientation to the EGFP gene. We found that reverse-transcribed spliced EGFP DNA sequences are generated in sperm cells and transmitted to embryos in IVF assays. After implantation in foster mothers, embryos developed into mice that expressed EGFP in the blood vessel endothelia of a variety of organs. The EGFP-encoding cDNA sequences were detected in positive tissues as extrachromosomal mosaic-propagated structures, maintained in low-copy number (< 1 copy/genome), and mosaic transmitted from founders to the F1 progeny. These results indicate that an efficient machinery is present in mature spermatozoa, which can transcribe, splice, and reverse-transcribe exogenous DNA molecules. This mechanism is implicated in the genesis and non-Mendelian propagation of new genetic information besides that contained in chromosomes

Exposure of normal and transformed cells to nevirapine, a reverse transcriptase inhibitor, reduces cell growth and promotes differentiation.

Endogenous, nontelomeric reverse transcriptase (RT) is encoded by two classes of repeated elements: retrotransposons and endogenous retroviruses. Expression of RT-coding genes is generally repressed in differentiated nonpathological tissues, yet is active in the mammalian germ line, embryonic tissues and tumor cells. Nevirapine is a non-nucleoside RT inhibitor with a well-characterized inhibitory activity on RT enzymes of retroviral origin. Here, we show that nevirapine is also an effective inhibitor of the endogenous RT in murine and human cell lines. In addition, progenitor and transformed cells undergo a significant reduction in the rate of cell growth upon exposure to nevirapine. This is accompanied by the onset of differentiation, as depicted in F9 and C2C7 progenitor cells cultures in which nevirapine triggers the expression of differentiation-specific markers. Consistent with this, an extensive reprogramming of cell cycle gene expression was depicted in nevirapine-treated F9 cultures. Furthermore, nevirapine exposure rescued the differentiation block present in acute myeloid leukemia (AML) cell lines and primary blasts from two AML patients, as indicated by morphological, functional and immunophenotypic assays. The finding that an RT inhibitor can modulate cell proliferation and differentiation suggests that RT may represent a novel target in the development of therapeutical approaches to neoplasia