Undifferentiated cells and embryos express high levels of endogenous non-telomerase reverse transcriptase (RT) of retroposon/retroviral origin. We previously found that RT inhibitors modulate cell growth and differentiation in several cell lines. We have now sought to establish whether high levels of RT activity are directly linked to cell transformation. To address this possibility, we have employed two different approaches to inhibit RT activity in melanoma and prostate carcinoma cell lines: pharmacological inhibition by two characterized RT inhibitors, nevirapine and efavirenz, and downregulation of expression of RT-encoding LINE-1 elements by RNA interference (RNAi). Both treatments reduced proliferation, induced morphological differentiation and reprogrammed gene expression. These features are reversible upon discontinuation of the anti-RT treatment, suggesting that RT contributes to an epigenetic level of control. Most importantly, inhibition of RT activity in vivo antagonized tumor growth in animal experiments. Moreover, pretreatment with RT inhibitors attenuated the tumorigenic phenotype of prostate carcinoma cells inoculated in nude mice. Based on these data, the endogenous RT can be regarded as an epigenetic regulator of cell differentiation and proliferation and may represent a novel target in cancer therapy.

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.

In higher eukaryotes, reverse transcriptase (RT) activities are encoded by a variety of endogenous retroviruses and retrotransposable elements. We previously found that mouse preimplantation embryos are endowed with an endogenous RT activity. Inhibition of that activity by the non nucleosidic inhibitor nevirapine or by microinjection of anti-RT antibody caused early embryonic developmental arrest. Those experiments indicated that RT is required for early development, but did not identify the responsible coding elements. We now show that microinjection of morpholino-modified antisense oligonucleotides targeting the 5' end region of active LINE-1 retrotransposons in murine zygotes irreversibly arrests preimplantation development at the two- and four-cell stages; the overall level of functional RT is concomitantly downregulated in arrested embryos. Furthermore, we show that the induction of embryo developmental arrest is associated with a substantial reprogramming of gene expression. Together, these results support the conclusion that expression of LINE-1 retrotransposons is required for early embryo preimplantation development.

Mature sperm cells, either of epididymal origin or ejaculated and depleted of seminal fluid, are spontaneously able to bind exogenous DNA molecules which are subsequently internalized into sperm nuclei. Southern blot analysis showed that the internalized DNA was specifically cleaved by sperm endonucleases and showed typical fragmentation patterns of localized hypersensitivity. Nucleases were activated in response to the internalization of exogenous DNA by sperm cells and their activity increased with the DNA concentration. Nuclease activation was efficient in epididymal sperm cells, while being drastically reduced in ejaculated washed spermatozoa. Nucleases were Ca++ dependent, and were, respectively, inhibited and activated by preincubating sperm cells with Aurintricarboxylic Acid (ATA) and Ca++ Ionophore A23187, which are known to, respectively, inhibit and activate apoptosis in somatic cells. Moreover, nuclease activation also caused a partial degradation of the sperm endogenous chromosomal DNA; cleaved DNA fragments were released from the sperm cells to the medium. Taken together, these results suggest that a metabolically active process similar to apoptosis is triggered in the nuclei of mature sperm cells upon interaction with exogenous DNA.

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.

We report that a reverse transcriptase (RT) activity is present in early cleavage stage embryos as determined by a Polymerase chain reaction (PCR)-based detection assay. In an attempt to establish whether this activity plays a role in early embryonic development, we have blocked the endogenous RT by two independent approaches: (1) embryos were exposed to nevirapine, a highly specific nonnucleoside inhibitor of RT activity; (2) anti-RT antibody was microinjected into the nucleus of one blastomere of 2-cell embryos. When embryos were exposed to nevirapine in the developmental window between late 1-cell and 4-cell stages, development was arrested before the blastocyst stage. In contrast, development was not affected when embryos were exposed to nevirapine after the eight-cell stage. Developmental arrest was also induced when anti-RT antibody was microinjected in one blastomere of 2-cell embryos. Analysis of gene expression by RT-PCR in nevirapine-arrested 2-cell embryos revealed an extensive and specific reprogramming of gene expression, involving both developmentally regulated and constitutively expressed genes, compared to control embryos. These results support the conclusion that an endogenous RT activity is required in mouse early embryogenesis specifically between the late 1-cell and the 4-cell stage.

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.

The murine Htf9-a/RanBP1and Htf9-c genes are divergently transcribed from a bidirectional promoter. The Htf9-a gene encodes the RanBP1 protein, a major partner of the Ran GTPase. The divergently transcribed Htf9-cgene encodes a protein sharing similarity with yeast and bacterial nucleic acid-modifying enzymes. We report here that both mRNA species produced by the Htf9-associated genes are regulated during the cell cycle progression, peak in S phase and decrease during mitosis. Transient expression experiments with reporter constructs showed that cell cycle expression is controlled at the transcriptional level, because the bidirectional Htf9 promoter is down-regulated in growth-arrested cells, is activated at the G1/S transition and reaches maximal activity in S phase, though with a different efficiency for each orientation. We have delimited specific promoter regions controlling S phase activity in one or both orientations: identified elements contain recognition sites for members belonging to both the E2F and Sp1 families of transcription factors. Together, the results suggest that the sharing of the regulatory region supports co-regulation of the Htf9-a/RanBP1 and Htf9-cgenes in a common window of the cell cycle.