Search

In the last fifty years, large efforts have been deployed in basic research, clinical oncology, and clinical trials, yielding an enormous amount of information regarding the molecular mechanisms of cancer and the design of effective therapies. The knowledge that has accumulated underpins the complexity, multifactoriality, and heterogeneity of cancer, disclosing novel landscapes in cancer biology with a key role of genome plasticity. Here, we propose that cancer onset and progression are determined by a stress-responsive epigenetic mechanism, resulting from the convergence of upregulation of

The notion that epigenetic information can be transmitted across generations is supported by mounting waves of data, but the underlying mechanisms remain elusive. Here, a model is proposed which combines different lines of experimental evidence. First, it has been shown that somatic tissues exposed to stressing stimuli release circulating RNA-containing extracellular vesicles; second, epididymal spermatozoa can take up, internalize and deliver the RNA-containing extracellular vesicles to oocytes at fertilization; third, early embryos can process RNA-based information. These elements constitute the building blocks upon which the model is built. The model proposes that a continuous stream of epigenetic information flows from parental somatic tissues to the developing embryos. The flow can cross the Weismann barrier, is mediated by circulating vesicles and epididymal spermatozoa, and has the potential to generate epigenetic traits that are then stably acquired in the offspring. In a broader perspective, it emerges that a natural ‘assembly line’ operates continuously, aiming at passing the parental epigenetic blueprint in growing embryos.

Emerging data indicate that the reverse transcriptase (RT) protein encoded by LINE-1 transposable elements is a promising cancer target. Nonnucleoside RT inhibitors, e.g. efavirenz (EFV) and SPV122.2, reduce proliferation and promote differentiation of cancer cells, concomitant with a global reprogramming of the transcription profile. Both inhibitors have therapeutic anticancer efficacy in animal models. Here we have sought to clarify the mechanisms of RT inhibitors in cancer cells. We report that exposure of PC3 metastatic prostate carcinoma cells to both RT inhibitors results in decreased proliferation, and concomitantly induces genome damage. This is associated with rearrangements of the nuclear architecture, particularly at peripheral chromatin, disruption of the nuclear lamina, and budding of micronuclei. These changes are reversible upon discontinuation of the RT-inhibitory treatment, with reconsititution of the lamina and resumption of the cancer cell original features. The use of pharmacological autophagy inhibitors proves that autophagy is largely responsible for the antiproliferative effect of RT inhibitors. These alterations are not induced in non-cancer cell lines exposed to RT inhibitors. These data provide novel insight in the molecular pathways targeted by RT inhibitors in cancer cells.

The active uptake of exogenous nucleic acids by spermatozoa of virtually all animal species is a well-established phenomenon whose significance has long been underappreciated. A growing body of published data demonstrates that extracellular vesicles released from mammalian somatic tissues pass an RNA-based flow of information to epididymal spermatozoa, thereby crossing the Weismann barrier. That information is delivered to oocytes at fertilization and affects the fate of the developing progeny. We propose that this essential process of epigenetic transmission depends upon the documented ability of epididymal spermatozoa to bind and internalize foreign nucleic acids in their nuclei. In other words, spermatozoa are not passive vectors of exogenous molecules but rather active participants in essential somatic communication across generations.

LINE-1 elements account for about 17% of the human genome and harbour two open reading frames: ORF1, encoding a 40 kDa RNA-binding protein, and ORF2, coding for a 150 kDa protein with reverse transcriptase (RT) activity. LINE-1s are highly expressed in embryos and tumor cells while being virtually silent in differentiated tissues and, consistently, both ORF-1p and ORF-2p have been detected in human cancers. RT-encoding ORF2 is expressed early in pre-neoplastic lesions suggesting that RT expression may be a potential cause, rather than a consequence, of cancer onset. Experimental data emerging from in vitro and in vivo studies confirm this view. Preclinical work showed that RT inhibition reduces proliferation, promotes differentiation of cancer cells and antagonizes tumor progression in murine models. Moreover, a recent phase II trial on metastatic hormone-resistant prostate cancer patients has confirmed the anticancer efficacy of RT inhibitors. Together, these data indicate that LINE-1-encoded RT emerges as a potential therapeutic target for a large spectrum of cancers and RT inhibitors as effective tools in a novel anti-cancer, non-cytotoxic, differentiation therapy.

LINE-1 (L1) retrotransposons are a source of endogenous reverse transcriptase (RT) activity, which is expressed as part of the L1-encoded ORF2 protein (L1-ORF2p). L1 elements are highly expressed in many cancer types but not in differentiated somatic tissues. We previously found that RT inhibition reduces cell proliferation and promote differentiation in neoplastic cells, indicating that high endogenous RT activity promote cancer growth. Here we investigate the expression of L1-ORF2p in several human types ocancer.We have developed a highly specific monoclonal antibody (mAb chA1-L1) raised against the endonuclease domain of L1-ORF2p as a tool to study ORF2p expression and localization in human cancer cell lines and staged bioptic tissues. We uncover new evidence for high levels of L1-ORF2p in transformed cell lines and staged epithelial cancer tissues (colon, prostate, lung and breast) while no or only basal ORF2p expression was detected in non-transformed cells. An in-depth analysis of colon and prostate cancer tissues shows ORF2p expression in pre-neoplastic stages, namely transitional mucosa and prostate intraepithelial neoplasia (PIN), respectively. Our results show that L1-ORF2p is overexpressed in tumor tissues and in pre-neoplastic colon and prostate tissues; this latter finding suggests that ORF2p could be considered as a potential early cancer diagnostic biomarker.

Long Interspersed Nuclear Elements (LINE-1) make up a large family of autonomous retrotransposons, accounting for about 17% of the human genome. They constitute the major source of non telomeric Reverse Transcriptase (RT), an essential component of the retrotransposition machinery. Expression of RT-encoding LINE-1 sequences is low in differentiated, non-pathological cells and highly active in early embryos, germ cells and in a broad spectrum of cancers. Growing evidence functionally implicate RT in control of cell growth and differentiation and suggest causative roles in cancer onset. Indeed, inhibition of RT activity reduces proliferation, promotes differentiation and antagonizes cancer progression in animal models. More recently, RT inhibition proved effective in a phase II clinical trial with metastatic prostate cancer patients. Furthermore, the LINE-1-encoded ORF2p product, ecompassing the RTencoding sequence, was found to be already esxpressed in precancerous lesions, increasing in progressive stages, while being undetectable in normal tissues. RT emerges therefore as a promising therapeutic target and a potential marker for early cancer detection. At the molecular level, the inhibition of LINE-1-encoded RT yields a global reprogramming of the gene expression profile in cancer cells, involving all RNA classes: coding mRNAs, long and small non-coding transcripts, including miRNAs - some of which are themselevs key players in cancer progression, invasion, and metastasis. In summary, the LINE-1-encoded RT emerges as a key component of a genome-wide regulatory mechanism that is active in embryogenesis, repressed during cell differentiation, and aberrantly reactivated in cancer cells.

LINE-1(longinterspersednuclearelements)retrotransposonsconstitutealargefamilyofretrotransposableelements, accounting for 17% of the human genome. They encode proteins required for their own mobilization, including a reverse transcriptase (RT) enzyme highly expressed in mouse embryos and mouse and human cancer cells and repressedinsomaticdifferentiatedhealthycells.Wehavefoundthatreversetranscriptiontakesplaceinearlymurine embryos, yielding an increase in LINE-1 copy number during preimplantation development, which also occurs in tumor progression. RT inhibition irreversibly arrests embryo development, reduces cancer cell proliferation, promotesdifferentiation,antagonizestumorgrowth,andcausesaglobalreprogrammingoftranscriptionprofiles.These resultsstronglysuggestthatapreviouslyunrecognizedRT-dependentregulatorymechanismoperatesduringpreimplantation development, is repressed during differentiation to normal tissues, and, when erroneously reactivated in adult life, promotes cell transformation and cancer progression by “resurrecting” embryonic transcriptional pathways.TheRT-dependentmechanismemergesasamajorsourceofgeneticandepigeneticchangeswithphysiological, pathological, and evolutionary implications.

Epigenetics is increasingly regarded as a potential contributing factor to evolution. Building on apparently unrelated results, here I propose that RNA-containing nanovesicles, predominantly small regulatory RNAs, are released from somatic tissues in the bloodstream, cross the Weismann barrier, reach the epididymis, and are eventually taken up by spermatozoa; henceforth the information is delivered to oocytes at fertilization. In the model, a LINE-1-encoded reverse transcriptase activity, present in spermatozoa and early embryos, plays a key role in amplifying and propagating these RNAs as extrachromosomal structures. It may be conceived that, over generations, the cumulative effects of sperm-delivered RNAs would cross a critical threshold and overcome the buffering capacity of embryos. As a whole, the process can promote the generation of an information-containing platform that drives the reshaping of the embryonic epigenetic landscape with the potential to generate ontogenic changes and redirect the evolutionary trajectory. Over time, evolutionary significant, stably acquired variations could be generated through the process. The interplay between these elements defines the concept of “evolutionary field”, a self-consistent, comprehensive information-containing platform and a source of discontinuous evolutionary novelty.

// Ilaria Sciamanna 1 , Alberto Gualtieri 1 , Pier Vincenzo Piazza 2 , Corrado Spadafora 1 1 Istituto Superiore di Sanita, Viale Regina Elena 299, Rome, Italy 2 NeuroCentre Magendie, INSERM U862, Univ Bdx2, Bordeaux, France Correspondence to: Corrado Spadafora, e-mail: cspadaf@tin.it Keywords: Retrotransposon, reverse transcriptase, inhibitor, non coding RNA, cancer therapy Received: August 01, 2014 Accepted: September 16, 2014 Published: October 29, 2014 ABSTRACT LINE-1 retrotransposons encode the reverse transcriptase (RT) enzyme, required for their own mobility, the expression of which is inhibited in differentiated tissues while being active in tumors. Experimental evidence indicate that the inhibition of LINE-1-derived RT restores differentiation in cancer cells, inhibits tumor progression and yields globally reprogrammed transcription profiles. Newly emerging data suggest that LINE-1-encoded RT modulates the biogenesis of miRNAs, by governing the balance between the production of regulatory double-stranded RNAs and RNA:DNA hybrid molecules, with a direct impact on global gene expression. Abnormally high RT activity unbalances the transcriptome in cancer cells, while RT inhibition restores ‘normal’ miRNA profiles and their regulatory networks. This RT-dependent mechanism can target the myriad of transcripts - both coding and non-coding, sense and antisense - in eukaryotic transcriptomes, with a profound impact on cell fates. LINE-1-encoded RT emerges therefore as a key regulator of a previously unrecognized mechanism in tumorigenesis

Epigenetics is increasingly regarded as a potential contributing factor to evolution. Building on apparently unrelated results, here I propose that RNA-containing nanovesicles, predominantly small regulatory RNAs, are released from somatic tissues in the bloodstream, cross the Weismann barrier, reach the epididymis, and are eventually taken up by spermatozoa; henceforth the information is delivered to oocytes at fertilization. In the model, a LINE-1-encoded reverse transcriptase activity, present in spermatozoa and early embryos, plays a key role in amplifying and propagating these RNAs as extrachromosomal structures. It may be conceived that, over generations, the cumulative effects of spermdelivered RNAs would cross a critical threshold and overcome the buffering capacity of embryos. As a whole, the process can promote the generation of an information-containing platform that drives the reshaping of the embryonic epigenetic landscape with the potential to generate ontogenic changes and redirect the evolutionary trajectory. Over time, evolutionary significant, stably acquired variations could be generated through the process. The interplay between these elements defines the concept of "evolutionary field", a self-consistent, comprehensive information-containing platform and a source of discontinuous evolutionary novelty.

Two large families of retrotransposons, that is, LINE-1 (Long Interspersed Nuclear Elements-1) and endogenous retroviruses, encode reverse transcriptase (RT) proteins in vertebrates. We previously showed that mouse preimplantation embryos are endowed with an endogenous, functional RT activity. Inhibiting that activity by microinjecting antisense oligonucleotides against a highly active LINE-1 family member in mouse oocytes blocked developmental progression between the two- and four-blastomere stages, indicating that LINE-1-encoded RT activity is strictly required at this critical transition in early development. Here we show that incubation of mouse zygotes with 5'-bromodeoxyuridine (BrdU) yields massive incorporation of this nucleoside analogue in newly synthesized DNA; surprisingly, a significant incorporation still occurs in both zygotic pronuclei in the presence of aphidicolin, a specific inhibitor of DNA replication. This aphidicolin-resistant BrdU incorporation is quantitatively abolished when embryos are simultaneously exposed to abacavir, a nucleoside RT inhibitor, indicating its retrotranscription-dependent nature. Moreover, quantitative PCR analysis revealed a burst of new synthesis of LINE-1 copies at the zygote- and two-cell embryo stages. These findings support the conclusion that RT-dependent amplification of LINE-1 retrotransposons is a distinctive feature of early embryonic genomes. Its physiological involvement in preimplantation murine development is discussed.

LINE-1 (Long Interspersed Nuclear Elements) and HERVs (Human Endogenous Retroviruses) are two families of autonomously replicating retrotransposons that together account for about 28% of the human genome. Genes harbored within LINE-1 and HERV retrotransposons, particularly those encoding the reverse transcriptase (RT) enzyme, are generally expressed at low levels in differentiated cells, but their expression is upregulated in transformed cells and embryonic tissues. Here we discuss a recently discovered RT-dependent mechanism that operates in tumorigenesis and reversibly modulates phenotypic and functional variations associated with tumor progression. Downregulation of active LINE-1 elements drastically reduces the tumorigenic potential of cancer cells, paralleled by reduced proliferation and increased differentiation. Pharmacological RT inhibitors (e.g., nevirapine and efavirenz) exert similar effects on tumorigenic cell lines, both in culture and in animal models. The HERV-K family play a distinct complementary role in stress-dependent transition of melanoma cells from an adherent, non-aggressive, to a non-adherent, highly malignant, growth phenotype. In synthesis, the retrotransposon-encoded RT is increasingly emerging as a key regulator of tumor progression and a promising target in a novel anti-cancer therapy.

This review summarizes emerging evidence that LINE-1 (Long Interspersed Nuclear Elements) -encoded reverse transcriptase (RT) regulates fundamental biological processes. Earlier studies showed that sperm cells can be used as vectors of both exogenous DNA and RNA molecules in sperm-mediated gene transfer assays. During these studies, a sperm endogenous RT activity was identified, which can reverse-transcribe exogenous RNA directly, or DNA molecules through sequential transcription and reverse transcription. Resulting cDNA copies generated in sperm cells can be delivered to embryos at fertilization, further propagated in tissues as low-copy extrachromosomal structures and transmitted to the progeny in a non-mendelian fashion. Being transcriptionally competent, they can induce phenotypic variations in positive tissues. An RT activity is also present in preimplantation embryos, and its inhibition causes developmental arrest in early preimplantation stages, paralleled by an extensive reprogramming of gene expression. In analogy with this, drug-mediated inhibition of RT activity, or RNA interference-mediated silencing of human LINE-1, reduce cell proliferation and induce differentiation in a variety of cancer cell lines. Furthermore, RT inhibition in vivo antagonizes the growth of human tumors in animal models. As a whole, these data implicate a RT-dependent machinery in the genesis of new genetic information in spermatozoa and in normal and pathological developmental processes.

In higher eukaryotic genomes, Long Interspersed Nuclear Element 1 (LINE-1) retrotransposons represent a large family of repeated genomic elements. They transpose using a reverse transcriptase (RT), which they encode as part of the ORF2p product. RT inhibition in cancer cells, either via RNA interference-dependent silencing of active LINE-1 elements, or using RT inhibitory drugs, reduces cancer cell proliferation, promotes their differentiation and antagonizes tumor progression in animal models. Indeed, the nonnucleoside RT inhibitor efavirenz has recently been tested in a phase II clinical trial with metastatic prostate cancer patients. An in-depth analysis of ORF2p in a mouse model of breast cancer showed ORF2p to be precociously expressed in precancerous lesions and highly abundant in advanced cancer stages, while being barely detectable in normal breast tissue, providing a rationale for the finding that RT-expressing tumours are therapeutically sensitive to RT inhibitors. We summarise mechanistic and gene profiling studies indicating that highly abundant LINE-1-derived RT can sequester RNA substrates for reverse transcription in tumor cells, entailing the formation of RNA:DNA hybrid molecules and impairing the overall production of regulatory miRNAs, with a global impact on the cell transcriptome. Based on these data, LINE-1-ORF2 encoded RT has a tumor-promoting potential that is exerted at an epigenetic level. We propose a model whereby LINE1-RT drives a previously unrecognized global regulatory process, the deregulation of which drives cell transformation and tumorigenesis and possibly implicated in cancer cell heterogeneity.

Mature spermatozoa are permeable to foreign DNA and RNA molecules. Here I propose a model, whereby extrachromosomal genetic information, mostly encoded in the form of RNA in somatic cells, can cross the Weismann barrier and reach epididymal spermatozoa. LINE-1 retrotransposon-derived reverse transcriptase (RT) can play key roles in the process by expanding the RNA-encoded information. Retrotransposon-encoded RT is stored in mature gametes, is highly expressed in early embryos and undifferentiated cells, and becomes downregulated in differentiated cells. In turn, RT plays a role in developmental control, as its inhibition arrests developmental progression of early embryos with globally altered transcriptomic profiles. Thus, sperm cells act as recipients, and transgenerational vectors of somatically derived genetic information which they pass to the next generation with the potential to modify the fate of the developing embryos.

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.

Two classes of repeated genomic elements, retrotransposons and endogenous retroviruses, encode for endogenous nontelomeric reverse transcriptase (RT), a gene that is down-regulated in differentiated cells but is highly expressed in embryonic and transformed tissues. Two nonnucleosidic RT inhibitors, efavirenz and nevirapine, currently used in HIV treatment, reversibly down-regulate tumor growth and induce differentiation in several human tumor cell models.Aggressive biological behavior and loss of specific thyroid cell functions, such as thyroglobulin, thyroid peroxidase, TSH receptor, Na/I symporter expression, and iodine uptake are features of anaplastic thyroid cancer. Thus, we evaluated the use of RT inhibitors as a potentially differentiating and molecular-targeted treatment of this neoplasm.Our findings showed that nevirapine and efavirenz reversibly inhibit cell proliferation without triggering cell death in the undifferentiated thyroid carcinoma ARO and FRO cells, which exhibited high levels of endogenous RT activity. Inhibition of cell growth was correlated with accumulation of cells in the G0/G1 phase of the cell cycle, with a concomitant decrease in the S phase. Moreover, treated cells demonstrated a differentiated phenotype and a significant reprogramming of gene expression characterized by up-regulation of the TSH receptor, thyroglobulin, thyroid peroxidase, and Na/I symporter genes. Interestingly, RT inhibition reestablished the ability to uptake iodine in response to TSH either in vitro or in vivo and reversibly down-regulated tumor growth in mice xenografts of ARO cells.These findings support the need for clinical trials to clarify whether RT inhibitors may restore the sensitivity to radiometabolic therapy in anaplastic thyroid tumors.

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.

Recent developments in studies of sperm-mediated gene transfer (SMGT) now provide solid ground for the notion that sperm cells can act as vectors for exogenous genetic sequences. A substantive body of evidence indicates that SMGT is potentially useable in animal transgenesis, but also suggests that the final fate of the exogenous sequences transferred by sperm is not always predictable. The analysis of SMGT-derived offspring has shown the existence of integrated foreign sequences in some cases, while in others stable modifications of the genome are difficult to detect. The appearance of SMGT-derived modified offspring on the one hand and, on the other hand, the rarity of actual modification of the genome, suggest inheritance as extrachromosomal structures. Several specific factors have been identified that mediate distinct steps in SMGT. Among those, a prominent role is played by an endogenous reverse transcriptase of retrotransposon origin. Mature spermatozoa are naturally protected against the intrusion of foreign nucleic acid molecules; however, particular environmental conditions, such as those occurring during human assisted reproduction, can abolish this protection. The possibility that sperm cells under these conditions carry genetic sequences affecting the integrity or identity of the host genome should be critically considered. These considerations further suggest the possibility that SMGT events may occasionally take place in nature, with profound implications for evolutionary processes.

Exogenous DNA molecules are spontaneously taken up by sperm cells, internalized in nuclei, and eventually integrated in the sperm genome. The actual occurrence of the integration suggests that the sperm chromosomal DNA is not uniformly and tightly packed with protamines, implying the existence of genomic sites where the chromosomal DNA is accessible to foreign molecules. We have characterized a hypersensitive, nucleosomal subfraction of mouse sperm chromatin that is highly enriched in unmethylated retroposon DNA from a variety of families. Here we propose that both the integration of exogenous DNA molecules, and the endogenous retroposition activity, occur in the same site(s) of sperm chromatin.

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.

Summary Sperm cells from a variety of species share the spontaneous ability to take up foreign DNA. That feature has been exploited to generate genetically modified animals with variable efficiency in different species. An unexpectedly large set of factors appears to modulate the interaction of sperm cells with exogenous DNA. The binding is mediated by specific DNA-binding proteins and is antagonized by an inhibitory factor in the seminal fluid. A portion of sperm-bound DNA is internalized in nuclei, a process mediated by CD4 molecules. Sperm interaction with foreign DNA triggers endogenous nuclease(s) that cleaves both the exogenous and the genomic DNA, eventually leading to a cell death process which resembles apoptosis. Internalized foreign DNA sequences reach the nuclear matrix and undergo recombination with chromosomal DNA. From these studies, a surprising network of metabolic functions is beginning to emerge in mature spermatozoa, which are normally repressed and are specifically activated upon exposure to appropriate stimuli. BioEssays 20:955‐964, 1998. r 1998 John Wiley & Sons, Inc.

Exposure of spermatozoa to stress conditions causes a drastic reduction of their fertilizing ability. We report here that the decrease in fertilization can be effectively antagonized by preincubating sperm cells with the nuclease inhibitor drug aurintricarboxylic acid (ATA). Preincubation of mouse epididymal sperm cells with ATA increased the yield of 2-cell embryos produced by in vitro fertilization assays. The effect of ATA was selectively exerted via spermatozoa, since neither preincubation of eggs, nor the direct treatment of zygotes, modified the yield of 2-cell-stage embryos. Our results suggest that ATA does not directly improve the ability of sperm cells to penetrate the egg cytoplasm but instead acts by preserving sperm nuclei from induced or spontaneously occurring damage and/or favors events that trigger early embryogenesis.

Sperm-mediated DNA transfer to offspring has the potential to markedly simplify the generation of transgenic animals, but the efficiency in mice has been controversial. To determine the basis of the variability of the procedure in mice, we undertook a large, collaborative study of sperm-mediated DNA transfer to mouse eggs in well-established laboratory conditions for in vitro fertilization and offspring development following embryo transfer. Sperm were incubated with plasmid DNA during the capacitation period and then added to freshly ovulated mouse oocytes for fertilization; cleaved embryos were then transferred to the oviducts of pseudopregnant recipients for gestation. From a total of 75 experiments, 13 produced 130 transgenic offspring, amounting to 7.4% of total fetuses. In five experiments, more than 85% of offspring were transgenic, but the factors leading to this high success rate were not discovered. Clustering of such a low frequency event could account for the disparate reports of transgenic success with sperm-mediated DNA transfer to mouse offspring. Discovering the factors important to success would not only allow this simplified approach to become an important tool in the generation of transgenic mice, but could also lead to important insights into natural protective mechanisms against sperm-mediated transfer of foreign DNA.

Mendelian laws provide the universal founding paradigm for the mechanism of genetic inheritance through which characters are segregated and assorted. In recent years, however, parallel with the rapid growth of epigenetic studies, cases of inheritance deviating from Mendelian patterns have emerged. Growing studies underscore phenotypic variations and increased risk of pathologies that are transgenerationally inherited in a non-Mendelian fashion in the absence of any classically identifiable mutation or predisposing genetic lesion in the genome of individuals who develop the disease. Non-Mendelian inheritance is most often transmitted through the germline in consequence of primary events occurring in somatic cells, implying soma-to-germline transmission of information. While studies of sperm cells suggest that epigenetic variations can potentially underlie phenotypic alterations across generations, no instance of transmission of DNA- or RNA-mediated information from somatic to germ cells has been reported as yet. To address these issues, we have now generated a mouse model xenografted with human melanoma cells stably expressing EGFP-encoding plasmid. We find that EGFP RNA is released from the xenografted human cells into the bloodstream and eventually in spermatozoa of the mice. Tumor-released EGFP RNA is associated with an extracellular fraction processed for exosome purification and expressing exosomal markers, in all steps of the process, from the xenografted cancer cells to the spermatozoa of the recipient animals, strongly suggesting that exosomes are the carriers of a flow of information from somatic cells to gametes. Together, these results indicate that somatic RNA is transferred to sperm cells, which can therefore act as the final recipients of somatic cell-derived information.

Mouse epididymal sperm cells have the spontaneous ability to take up exogenous DNA. A proportion of the sperm-bound DNA is further internalized into sperm nuclei. In this work, we have followed up the fate of the foreign DNA upon internalization into nuclei. We have found that the internalized plasmid DNA becomes tightly associated with the nuclear scaffold, is extensively rearranged, and undergoes recombination with the sperm genomic DNA. Sequence analysis of two randomly selected clones independently recovered by plasmid rescue from pSV2CAT plasmid-challenged sperm cells shows that DNA fragments from the plasmid are integrated into the mouse sperm genome. The sites of integration are identical in both clones, suggesting that these events do not occur randomly, but take place at preferential sites. A topoisomerase II consensus sequence is found adjacent to one end of the integration site, suggesting a possible role of this enzyme in the process of nonhomologous recombination.

Two 2,6-difluoro-DABO derivatives (MC 1047, 1, and MC 1220, 2, respectively) were tested against endogenous, nontelomeric reverse transcriptase (endo-RT) in human differentiating cell systems to investigate their antiproliferative and cytodifferentiating activity. The two compounds significantly reduced cell proliferation and facilitated the morphological differentiation of cells. These results propose F(2)-DABOs as useful tools in preventive and/or curative therapy to counteract the loss of differentiation in dedifferentiating pathologies and as antiproliferative drugs in tumor therapy.

Ejaculated sperm cells from both bovine and swine animals depleted of the seminal fluid were used as vectors for transferring different plasmid DNAs into eggs during the fertilization process. PCR screening of about 2000 blastocysts showed that transformed embryos were obtained in both animal species with varying efficiencies, and that the rate of transformation depended on the plasmid used. The most efficient rate was obtained in both species with the pSV2CAT construct, yielding 22% positive blastocysts in bovine and 5.7% in swine respectively. Artificial insemination of ten sows with boar sperm cells that had been preincubated with pSV2CAT DNA produced 82 offspring. Southern blot analysis of the DNAs extracted from the animal tails showed that five animals were transgenic and contained sequences complementary to pSV2CAT DNA that appeared to be rearranged compared to the original plasmid. These results indicate that sperm‐mediated DNA transfer protocols can be successfully adapted for the genera...

// Alberto Gualtieri 1 , Federica Andreola 2 , Ilaria Sciamanna 1 , Paola Sinibaldi-Vallebona 3 , Annalucia Serafino 2 and Corrado Spadafora 1 1 Istituto Superiore di Sanita, Rome, Italy; 2 Institute of Translational Pharmacology, CNR, Rome, Italy; 3 Department of Experimental Medicine and Surgery - University of Rome “Tor Vergata”, Rome, Italy. Correspondence: Corrado Spadafora, email: // Keywords : LINE-1 retrotransposons; SINE retrotransposons; reverse transcriptase; copy number amplification; breast cancer progression Received : July 17, 2013 Accepted : August 7, 2013 Published : August 9, 2013 Abstract In higher eukaryotic genomes, Long Interspersed Nuclear Element 1 (LINE-1) retrotransposons and endogenous retroviruses represent large families of repeated elements encoding reverse transcriptase (RT) proteins. Short Interspersed Nuclear Element B1 (SINE B1) retrotrasposons do not encode RT, but use LINE-1-derived RT for their retrotransposition. We previously showed that many cancer types have an abundant endogenous RT activity. Inhibition of that activity, by either RNA interference-dependent silencing of active LINE-1 elements or by RT inhibitory drugs, reduced proliferation and promoted differentiation in cancer cells, indicating that LINE-1-encoded RT is required for tumor progression. Using MMTV-PyVT transgenic mice as a well-defined model of breast cancer progression, we now report that both LINE-1 and SINE B1 retrotransposons are up-regulated at a very early stage of tumorigenesis; LINE-1-encoded RT product and enzymatic activity were detected in tumor tissues as early as stage 1, preceding the widespread appearance of histological alterations and specific cancer markers, and further increased in later progression stages, while neither was present in non-pathological breast tissues. Importantly, both LINE-1 and SINE B1 retrotransposon families undergo copy number amplification during tumor progression. These findings therefore indicate that RT activity is distinctive of breast cancer cells and that, furthermore, LINE-1 and SINE B1 undergo copy number amplification during cancer progression.

Background: Malignant melanoma is one of the most aggressive types of skin cancers and its etiology is not yet clear. Phenotypic-switching has been associated with melanoma tumor aggressiveness and metastasis. Microenvironmental factors such as estrogen, cytokines and other stress conditions determine epigenetic events, as hypo-methylation, which are involved in cell transformation. Interestingly, endogenous retroviruses contribute to melanoma progression and are transcriptionally regulated by epigenetic events. Previously, we demonstrated that the aggressiveness and immune evasion of metastatic melanoma is partly depends on human endogenous retrovirus-K (HERV-K) activation. Thus, studying the role of HERV-K activation in melanoma phenotypic-switching is important to understand melanomagenesis and find possible therapeutic targets. Objective: To investigate the potential role of HERV-K activation in melanoma cells phenotypic-switching in response to change in microenvironment conditions. Methods: the TVM-A12 cell line, isolated in our laboratory from a metastatic melanoma lesion, and other commercial melanoma cell lines (WM-266-4, WM-115 and A375) were cultured in standard, differentiation and serum-free cell culture media. RNA interference, flow cytometry, qRT-PCR, self-renewing assay, sphere-forming assay and migration/invasion assays were performed. Data were analyzed using SPSS software version 17. Results: TVM-A12 showed high cellular plasticity as capable to acquire different phenotypes depending on the modification of the microenvironment. Indeed, in differentiation media the TVM-A12 modified the morphology towards a more differentiated phenotype, but, surprisingly, the cells were not committed to a final stage of differentiation and were able to revert to the original phenotype. The exposure of TVM-A12 to a serum-free medium, induced changes in cell growth and morphology, from adherent towards sphere-like cellular aggregates, characterized by an increased activation of HERV-K expression and generation of a CD133+ subpopulation of melanoma cells. Under this stress-condition, the silencing of HERV-K expression in TVM-A12 cells by RNA interference, significantly abolished the generation of the CD133+ subpopulation, dysregulated the cellular aggregates phenotype and suppressed their proliferation. More appreciably, the induction of HERV-K expression and the stress-mediated generation of CD133+ subpopulation of melanoma cells were also seen in other primary and metastatic melanoma cell lines like WM-115, WM-266-4 and A375 with a considerable variability. Furthermore, theTVM-A12-CD133+ cells, sorted from TVM-A12, showed dynamic cell plasticity upon the modification of the microenvironments and displayed a significantly higher self-renewing, migration and invasion capacity than the parental TVM-A12 cells. Moreover, treatment of TVM-A12 and TVM-A12-CD133+ with the non-nucleoside reverse transcriptase inhibitors (NNRTIs), nevirapine and efavirenz, inhibited the expression of HERV-K and significantly induced high levels of apoptosis in TVM-A12-CD133+ cells. Conclusions: these results demonstrated for the first time that HERV-K has a decisive role on phenotype-switching in metastatic melanoma cells, and the generation of the more aggressive CD133+ subpopulation in stressful microenvironments. Moreover, NNRTIs treatments were able to inhibit the expression of HERV-K and affect the stress-mediated generation of the CD133+ subpopulation. Thus, the further understanding of the dynamics of HERV-K in melanoma will help to understand melanomagenesis and find possible new therapeutic targets. Citation Format: Ayele Argaw-Denboba, Emanuela Balestrieri, Annalucia Serafino, Ilaria Bucci, Chiara Cipriani, Roberta Sorrentino, Corrado Spadafora, Paola Sinibaldi-Vallebona, Claudia Matteucci. Role of human endogenous retrovirus-K in phenotype-switching of metastatic melanoma cells during microenvironment alterations. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C03.

Mature sperm cells have the spontaneous capability of taking up exogenous DNA. Potential substrates for the interaction of the DNA with the sperm heads are specific classes of DNA-binding proteins. In the present work three major classes of DNA-binding proteins were identified by Southwestern analysis of sperm head protein extracts: a first class of about 50 kDa in molecular weight, a second one of 30-35 kDa, and finally a third one below 20 kDa. The latter group most probably contains sperm protamines. Our attention was particularly focused on the 30- to 35-kDa proteins as a substrate for DNA binding, as they represented the only group whose electrophoretic mobility was conserved among mammalian species. In addition they were the only class of DNA-binding proteins accessible to exogenous DNA in intact sperm cells. The purified 30- to 35-kDa proteins interacted in vitro with exogenous DNA and generated discrete protein/DNA complexes as determined by band shift assay. A factor blocking the binding of exogenous DNA to sperm cells was also identified in the seminal fluid of mammals and in echinoid spermatozoa. The factor also exerted a powerful inhibitory effect on DNA uptake in sperm cells of heterologous species. The 30- to 35-kDa DNA-binding proteins appeared to be the specific target through which the inhibition was mediated. In the presence of the inhibitory factor, the 30- to 35-kDa lost the ability to bind exogenous DNA. Thus, the interaction of exogenous DNA with sperm cells does not appear to be a casual event but, on the contrary, relies on a molecular mechanism based on the cooperation of specific protein factors.

Two large families of retrotransposons, that is, LINE-1 (Long Interspersed Nuclear Elements-1) and endogenous retroviruses, encode reverse transcriptase (RT) proteins in vertebrates. We previously showed that mouse preimplantation embryos are endowed with an endogenous, functional RT activity. Inhibiting that activity by microinjecting antisense oligonucleotides against a highly active LINE-1 family member in mouse oocytes blocked developmental progression between the two- and four-blastomere stages, indicating that LINE-1-encoded RT activity is strictly required at this critical transition in early development. Here we show that incubation of mouse zygotes with 5'-bromodeoxyuridine (BrdU) yields massive incorporation of this nucleoside analogue in newly synthesized DNA; surprisingly, a significant incorporation still occurs in both zygotic pronuclei in the presence of aphidicolin, a specific inhibitor of DNA replication. This aphidicolin-resistant BrdU incorporation is quantitatively abolished when embryos are simultaneously exposed to abacavir, a nucleoside RT inhibitor, indicating its retrotranscription-dependent nature. Moreover, quantitative PCR analysis revealed a burst of new synthesis of LINE-1 copies at the zygote- and two-cell embryo stages. These findings support the conclusion that RT-dependent amplification of LINE-1 retrotransposons is a distinctive feature of early embryonic genomes. Its physiological involvement in preimplantation murine development is discussed.

LINE-1 (Long Interspersed Nuclear elements) and HERVs (Human Endogenous Retroviruses) are two families of retrotransposons which together account for about 28% of the human genome. Genes harbored within LINE-1 and HERV retrotransposons, particularly that encoding the reverse transcriptase (RT) enzyme, are generally expressed at low levels in differentiated cells, but their expression is up-regulated in embryonic tissues and transformed cells. Here we review evidence indicating that the LINE-1-encoded RT plays regulatory roles in early embryonic development. Indeed, antisense-mediated inhibition of expression of a highly expressed LINE-1 family in mouse zygotes caused developmental arrest at the two- or four-cell embryo stages. Development is also arrested when the embryo endogenous RT activity is pharmacologically inhibited by nevirapine, an RT inhibitor currently employed in AIDS treatment. The arrest of embryonic development is irreversible even after RT inhibition is removed and it is associated with subverted gene expression profiles. These data indicate an early requirement for LINE-1-encoded RT to support early developmental progression. Consistent with this, recent findings indicate that a reverse transcription wave is triggered in the zygote a few hours after fertilization and is propagated at least through the first two rounds of cell division. On the whole these findings suggest that reverse transcription is strictly required in early embryos as a key component of a novel RT-dependent mechanism that regulated the proper unfolding of the developmental program.

A series of 5-alkyl-2-(alkylthio)-6-(1-(2,6-difluorophenyl)propyl)-3,4-dihydropyrimidin-4(3H)-one derivatives (3a-h) belonging to the F(2)-DABOs class of non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) are endowed with a strong antiproliferative effect and induce cytodifferentiation in A375 melanoma cells. Among tested compounds, the most potent is 3g (SPV122), which also induces apoptosis in a cell-density-dependent manner and antagonizes tumor growth in animal models. All these effects are similar or even more pronounced than those previously reported for other nucleoside or non-nucleoside inhibitors of reverse transcriptase or by functional knockout of the reverse-transcriptase-encoding long interspersed element 1 by RNA interference (RNAi). Taken together with our previously reported results, these data further confirm our idea that cellular alterations induced by NNRTIs are a consequence of the inhibition of the endogenous reverse transcriptase in A375 cells and support the potential of NNRTIs as valuable agents in cancer therapy.

Epididymal sperm cells, incubated with plasmid DNA, showed a spontaneous tendency to interact with the exogenous nucleic acid. We have investigated the molecular basis of such interaction. Exogenous DNA is taken up by sperm cells over a 15- to 20-min period and is specifically localized on the nuclear area of the sperm head. DNA was reversibly bound to spermatozoa since it can be competed out by excess of cold competitor DNA or by other polyanions as heparin and dextran sulphate. By contrast, poly-L-lysine, a polycation, favours the uptake. DNA molecules of large size (7 kb) were preferentially taken up as compared to smaller ones (150-750 bp). Acidic proteins were also taken up and concentrated, as for DNA, at the nuclear level. These data strongly suggested that ionic interactions may occur between foreign molecules and a substrate located in the sperm head. On the basis of Southwestern analysis, a sperm head protein(s) of 30-35 KD is identified as potential substrate for exogenous DNA binding. Moreover, we have found that seminal plasma contains factor(s) which abolish sperm permeability, exerting a powerful inhibitor effect on DNA uptake. The presence of a specific binding protein for the DNA and of a factor inhibiting such interaction support the existence of a mechanism controlling, through specific factors, the sperm-DNA interaction.

Melanoma development is a multi-step process arising from a series of genetic and epigenetic events. Although the sequential stages involved in progression from melanocytes to malignant melanoma are clearly defined, our current understanding of the mechanisms leading to melanoma onset is still incomplete. Growing evidence show that the activation of endogenous retroviral sequences might be involved in transformation of melanocytes as well as in the increased ability of melanoma cells to escape immune surveillance. Here we show that human melanoma cells in vitro undergo a transition from adherent to a more malignant, non-adherent phenotype when exposed to stress conditions. Melanoma-derived non-adherent cells are characterized by an increased proliferative potential and a decreased expression of both HLA class I molecules and Melan-A/MART-1 antigen, similarly to highly malignant cells. These phenotypic and functional modifications are accompanied by the activation of human endogenous retrovirus K expression (HERV-K) and massive production of viral-like particles. Down-regulation of HERV-K expression by RNA interference prevents the transition from the adherent to the non-adherent growth phenotype in low serum. These results implicate HERV-K in at least some critical steps of melanoma progression.

Adaptation to hypoxia through activation of the hypoxia inducible factor-1 (HIF-1) is crucial for tumor cells survival. Here we describe the antitumoral effects of the new molecule CR 3294 on tumor cells in the presence of hypoxia. Treatment of the breast carcinoma cell line MDA-MB-231 with CR 3294 in 1% O(2) resulted in an in vivo and in vitro inhibition of tumor growth. CR 3294 induced accumulation of autophagosomes in hypoxic MDA-MB-231 cells as assessed by both transmission electron microscopy (TEM) and the autophagic marker LC3-II. TEM analysis revealed the presence of invaginations of the cytoplasm into the nucleus. Autophagosomes were present in such invaginations. Moreover, CR 3294 inhibited both the DNA binding of HIF-1alpha and VEGF mRNA synthesis. Immunoprecipitation and immunofluorescence studies showed an interaction between LC3 and HIF-1alpha. We next detailed the effect of inhibitors and activators of autophagy on both HIF-1alpha and LC3. In particular, 3 methyladenine (3MA) and wortmannin, two macroautophagic inhibitors, prevented both the decrease of HIF-1alpha protein levels and LC3 processing in cells treated with CR 3294. Bafilomycin and leupeptin, inhibitors of lysosomes, prevented HIF-1alpha decrease without affecting LC3 processing. By contrast, treating hypoxic MDA-MB-231 cells with trifluoperazine (TFP) or serum withdrawal (SW), two activators of autophagy, diminished HIF-1alpha levels and stimulated LC3 processing. These results indicate that activation of the autophagic pathway in hypoxic cells by the new molecule CR 3294, as well as by TFP or SW, can have potentially important implications for cancer treatment.

Sperm-mediated gene transfer (SMGT) is a procedure through which new genetic traits are introduced in animals by exploiting the ability of spermatozoa to take up exogenous DNA molecules and deliver them to oocytes at fertilization. The interaction of exogenous DNA with sperm cells is a regulated process mediated by specific factors; among those, a reverse transcriptase (RT) activity plays a central role in SMGT. 'Retro-genes' are generated either through reverse transcription of exogenous RNA internalized in spermatozoa, or through sequential transcription, splicing and reverse transcription of exogenous DNA. The resulting retro-genes are delivered to oocytes and transmitted to embryos and born animals as low-copy, transcriptionally competent, extrachromosomal structures capable of determining new phenotypic traits. Retro-genes can be further transmitted through sexual reproduction from founders to their F1 progeny: new genetic and phenotypic features, unlinked to chromosomes, can thus be generated and inherited in a non-Mendelian ratio. We have called this phenomenon sperm-mediated 'reverse' gene transfer (SMRGT). Thus, a RT-mediated machinery operates in sperm cells and is responsible for the genesis and non-Mendelian propagation of new genetic information. The features of RT-generated traits elicited in SMRGT resemble those characterized in recent studies of RNA-mediated inheritance of extra-genomic information.

Reverse Transcriptase (RT) activity is historically associated with the replication of infectious retroviruses. Cellular RT-coding genes have subsequently been identified in eukaryotic genomes. These genes are harbored within retrotransposable elements (retrotransposons and endogenous retroviruses), mobile DNA sequences characterized by the ability to integrate in mammalian genomes through RNA intermediates. Retrotransposition is mediated by an RT activity that catalyzes the reverse transcription of RNA into cDNA copies. A vast body of correlative evidence links up-regulated RT activity to cell systems with a high proliferative potential and low differentiation level, including embryonic tissues and tumors. In contrast, RT is silenced, or expressed at low levels, in differentiated cells. In recent work, we have used non-nucleosidic RT inhibitors widely employed to treat HIV infection and we have observed that these molecules exert a powerful cytostatic and differentiating activity in several models of human cancers both in vitro and in vivo, associated with the inhibition of endogenous RT activity. This review addresses the potential role of RT inhibitors as new anticancer therapeutic drugs. Based on preclinical observations, we also discuss the working hypothesis that the differentiating activity of RT inhibitors may re-establish or improve the efficacy of conventional treatments in specific conditions, such as hormone-refractory prostate carcinoma, anaplastic thyroid tumors and hematological malignancies. These novel findings strongly support the need for clinical trials to test the anti-tumor activity of RT inhibitors in specific malignancies.

In an article published in 1989,1 we reported that mouse epididymal spermatozoa take up exogenous DNA spontaneously and that the sperm-bound DNA molecules are subsequently transferred to embryos in IVF assays and further propagated to newborn mice. Despite initial controversy over the reproducibilit...

Transformed cells express high levels of non-telomeric reverse-transcriptase (RT) activity of retrotransposon and endogenous retrovirus origin. We previously reported that RT inhibition, either pharmacological or through transient silencing of RT-encoding LINE-1 (L1) elements by RNA interference (RNAi), reduced proliferation, induced differentiation and reprogrammed gene expression in human tumorigenic cell lines. Moreover, the antiretroviral drug efavirenz antagonized tumor progression in animal models in vivo. To get insight into the role of retroelements in tumorigenesis, we have now produced two cell lines derived from A-375 melanoma, in which the expression of either L1 retrotransposon, or HERV-K endogenous retrovirus, was stably suppressed by RNAi. Compared to the parental A-375 cell line, cells with stably interfered L1 expression show a lower proliferation rate, a differentiated morphology and lower tumorigenicity when inoculated in nude mice. L1 silencing modulates expression of several genes and, unexpectedly, also downregulates HERV-K expression. In HERV-K interfered cells, instead, L1 expression was unaffected, and cell proliferation and differentiation remained unchanged compared to parental A-375 cells. In vivo, however, their tumorigenic potential was found to be reduced after inoculation in nude mice. These results suggest that L1 and HERV-K play specific and distinct roles in cell transformation and tumor progression.

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 (