An Epigenetic LINE-1-Based Mechanism in Cancer.

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 LINE-1 (long interspersed nuclear element 1), the largest family of human retrotransposons, genome damage, nuclear lamina fragmentation, chromatin remodeling, genome reprogramming, and autophagy activation. The upregulated expression of LINE-1 retrotransposons and their protein products plays a key role in these processes, yielding an increased plasticity of the nuclear architecture with the ensuing reprogramming of global gene expression, including the reactivation of embryonic transcription profiles. Cancer phenotypes would thus emerge as a consequence of the unscheduled reactivation of embryonic gene expression patterns in an inappropriate context, triggering de-differentiation and aberrant proliferation in differentiated cells. Depending on the intensity of the stressing stimuli and the level of LINE-1 response, diverse degrees of malignity would be generated.

The active role of spermatozoa in transgenerational inheritance.

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 retrotransposon copies are amplified during murine early embryo development.

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.

Reverse transcriptase inhibitors promote the remodelling of nuclear architecture and induce autophagy in prostate cancer cells.

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.

Endogenous reverse transcriptase as a mediator of ursolic acid's anti-proliferative and differentiating effects in human cancer cell lines.

Ursolic acid (UA) is a pentacyclic triterpenoid compound that is widely distributed in the plant kingdom and has a broad range of biological effects. Here, we examined the effects of UA on the proliferation and differentiation of human tumor cell lines from melanoma (A375), glioblastoma (U87) and thyroid anaplastic carcinoma (ARO), and on the proliferation of a non-transformed human fibroblast cell line (WI-38). The results show that UA inhibits tumor cell proliferation in a dose- and time-dependent manner. Consistent with this finding, UA treatment promotes differentiation of all of the analyzed tumor cell lines. Interestingly, we found that UA inhibits the endogenous reverse transcriptase (RT) activity in tumor cells, which has recently been shown to be involved in the control of proliferation and differentiation of neoplastic cells. Considering these findings, we suggest that the observed anti-proliferative and differentiating effects of UA may be related to this target.

LINE-1-encoded reverse Transcriptase as a target in cancer therapy.

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.

Long interspersed nuclear element-1 expression and retrotransposition in prostate cancer cells.

BACKGROUND: Long Interspersed Nuclear Element-1 (LINE-1) is an autonomous retrotransposon that generates new genomic insertions through the retrotransposition of a RNA intermediate. Expression of LINE-1 is tightly repressed in most somatic tissues to prevent DNA damage and ensure genomic integrity. However, the reactivation of LINE-1 has been documented in cancer and the role of LINE-1 protein expression and retrotransposition has become of interest in the development, progression, and adaptation of many epithelial neoplasms, including prostate cancer. RESULTS: Here, we examined endogenous LINE-1 protein expression and localization in a panel of prostate cancer cells and observed a diverse range of LINE-1 expression patterns between cell lines. Subcellular localization of LINE-1 proteins, ORF1p and ORF2p, revealed distinct expression patterns. ORF1p, a nucleic acid chaperone that binds LINE-1 mRNA, was predominantly expressed in the cytoplasm, with minor localization in the nucleus. ORF2p, containing endonuclease and reverse transcriptase domains, exhibited punctate foci in the nucleus and also displayed co-localization with PCNA and γH2AX. Using a retrotransposition reporter assay, we found variations in LINE-1 retrotransposition between cell lines. CONCLUSIONS: Overall, our findings reveal new insight into the expression and retrotransposition of LINE-1 in prostate cancer. The prostate cancer cells we investigated provide a unique model for investigating endogenous LINE-1 activity and provide a functional model for studying LINE-1 mechanisms in prostate cancer.

Antitumor effect of combination of the inhibitors of two new oncotargets: proton pumps and reverse transcriptase.

Tumor therapy needs new approaches in order to improve efficacy and reduce toxicity of the current treatments. The acidic microenvironment and the expression of high levels of endogenous non-telomerase reverse transcriptase (RT) are common features of malignant tumor cells. The anti-acidic proton pump inhibitor Lansoprazole (LAN) and the non-nucleoside RT inhibitor Efavirenz (EFV) have shown independent antitumor efficacy. LAN has shown to counteract drug tumor resistance. We tested the hypothesis that combination of LAN and EFV may improve the overall antitumor effects. We thus pretreated human metastatic melanoma cells with LAN and then with EFV, both in 2D and 3D spheroid models. We evaluated the treatment effect by proliferation and cell death/apoptosis assays in classical and in pulse administration experiments. The action of EFV was negatively affected by the tumor microenvironmental acidity, and LAN pretreatment overcame the problem. LAN potentiated the cytotoxicity of EFV to melanoma cells and, when administered during the drug interruption period, prevented the replacement of tumor cell growth.This study supports the implementation of the current therapies with combination of Proton Pumps and Reverse Transcriptase inhibitors.

HERV-K activation is strictly required to sustain CD133+ melanoma cells with stemness features.

BACKGROUND: Melanoma is a heterogeneous tumor in which phenotype-switching and CD133 marker have been associated with metastasis promotion and chemotherapy resistance. CD133 positive (CD133+) subpopulation has also been suggested as putative cancer stem cell (CSC) of melanoma tumor. Human endogenous retrovirus type K (HERV-K) has been described to be aberrantly activated during melanoma progression and implicated in the etiopathogenesis of disease. Earlier, we reported that stress-induced HERV-K activation promotes cell malignant transformation and reduces the immunogenicity of melanoma cells. Herein, we investigated the correlation between HERV-K and the CD133+ melanoma cells during microenvironmental modifications. METHODS: TVM-A12 cell line, isolated in our laboratory from a primary human melanoma lesion, and other commercial melanoma cell lines (G-361, WM-115, WM-266-4 and A375) were grown and maintained in the standard and stem cell media. RNA interference, Real-time PCR, flow cytometry analysis, self-renewal and migration/invasion assays were performed to characterize cell behavior and HERV-K expression. RESULTS: Melanoma cells, exposed to stem cell media, undergo phenotype-switching and expansion of CD133+ melanoma cells, concomitantly promoted by HERV-K activation. Notably, the sorted CD133+ subpopulation showed stemness features, characterized by higher self-renewal ability, embryonic genes expression, migration and invasion capacities compared to the parental cell line. RNA interference-mediated downregulation experiments showed that HERV-K has a decisive role to expand and maintain the CD133+ melanoma subpopulation during microenvironmental modifications. Similarly, non nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine were effective to restrain the activation of HERV-K in melanoma cells, to antagonize CD133+ subpopulation expansion and to induce selective high level apoptosis in CD133+ cells. CONCLUSIONS: HERV-K activation promotes melanoma cells phenotype-switching and is strictly required to expand and maintain the CD133+ melanoma cells with stemness features in response to microenvironmental modifications.

Inhibition of endogenous reverse transcriptase antagonizes human tumor growth.

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.

The Reverse Transcriptase Encoded by LINE-1 Retrotransposons in the Genesis, Progression, and Therapy of Cancer.

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 non-nucleoside 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 tumors are therapeutically sensitive to RT inhibitors. We summarize mechanistic and gene profiling studies indicating that 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 with possible implications for cancer cell heterogeneity.

Enhanced expression of LINE-1-encoded ORF2 protein in early stages of colon and prostate transformation.

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, while being silenced in most differentiated somatic tissues. We previously found that RT inhibition reduces cell proliferation and promotes differentiation in neoplastic cells, indicating that high endogenous RT activity promotes cancer growth. Here we investigate the expression of L1-ORF2p in several human types of cancer.We have developed a highly specific monoclonal antibody (mAb chA1-L1) to study ORF2p expression and localization in human cancer cells and 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 tissues shows ORF2p expression in preneoplastic stages, namely transitional mucosa and prostate intraepithelial neoplasia (PIN), respectively.Our results show that L1-ORF2p is overexpressed in tumor and in preneoplastic colon and prostate tissues; this latter finding suggests that ORF2p could be considered as a potential early diagnostic biomarker.

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.

Mouse early embryos obtained by natural breeding or in vitro fertilization display a differential sensitivity to extremely low-frequency electromagnetic fields.

We have investigated the sensitivity of pre-implantation embryos obtained by natural breeding (NB) or in vitro fertilization (IVF) to extremely low-frequency magnetic fields (ELF-MF). Fertilized eggs obtained by NB were removed from mothers 12h after mating and cultured in vitro for 5 days under continuous ELF-MF exposure (constant strength of 50Hz and various intensities, i.e. 60, 120 and 220 microT). Alternatively, zygotes obtained by IVF were subjected to ELF-MF exposure (50Hz, 60 microT), starting 12h after IVF for 5 days. We found that ELF-MF exposure causes a small yet significant (P<0.05) decrease in the survival rate of NB-derived embryos at the latest stages of pre-implantation development, i.e. the eight cell-to-blastocyst transition. In embryos exposed to the highest field intensity (220 microT), the effect became apparent somewhat earlier. When IVF-derived embryos were exposed to ELF-MF, the reduction in the rate of embryo survival was more pronounced and the difference from controls was more significant (P<0.01). Moreover, the decreased survival rate in IVF embryos became apparent as early as the first cleavage and persisted throughout pre-implantation. These results suggest that IVF-derived embryos are more sensitive than NB-generated embryos to ELF-MF, and that this sensitivity occurs earlier in development.

Regulatory roles of LINE-1-encoded reverse transcriptase in cancer onset and progression.

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.

Increased expression and copy number amplification of LINE-1 and SINE B1 retrotransposable elements in murine mammary carcinoma progression.

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.

A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines.

LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the 'normal' small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells.

A reverse transcriptase-dependent mechanism is essential for murine preimplantation development.

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.

Modulation of cell differentiation, proliferation, and tumor growth by dihydrobenzyloxopyrimidine non-nucleoside reverse transcriptase inhibitors.

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.

Retrotransposons, reverse transcriptase and the genesis of new genetic information.

Spermatozoa of virtually all species can take up exogenous DNA or RNA molecules and internalize them into nuclei. A sperm endogenous reverse transcriptase activity can reverse-transcribe the internalized molecules in cDNA copies: exogenous RNA is reverse-transcribed in a one-step reaction, whereas DNA is first transcribed into RNA and subsequently reverse-transcribed. In either case, the newly synthesized cDNAs are delivered from sperm cells to oocytes at fertilization and are further propagated throughout embryogenesis and in tissues of adult animals. The reverse-transcribed sequences are underrepresented (below 1 copy/genome), mosaic distributed in tissues of adult individuals, transmitted in a non-Mendelian fashion from founders to F1 progeny, transcriptionally competent, variably expressed in different tissues and temporally transient, as they progressively disappear in aged animals. Based on these features, the reverse-transcribed sequences behave as extrachromosomal, biologically active retrogenes and induce novel phenotypic traits in animals. This RT-dependent mechanism, presumably originating from LINE-1 retroelements, generates transcriptionally competent retrogenes in sperm cells. These data strengthen the emerging view of a novel transgenerational genetics as the source of a continuous flow of novel epigenetic and phenotypic traits, independent from those associated to chromosomes. The distinctive features of this retrotransposon-based phenomenon share analogies with a recently discovered form of RNA-mediated inheritance, compatible with a Lamarckian-type adaptation.