Ilaria Sciamanna

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.

Distinct roles for LINE-1 and HERV-K retroelements in cell proliferation, differentiation and tumor progression

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.

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.

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.

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.

DNA dose and sequence dependence in sperm-mediated gene transfer.

We have tested three parameters in sperm‐mediated gene transfer assays with mice and pigs: (i) the epididymal versus ejaculated origin of sperm cells, (ii) the primary structure, and (iii) the amount of the challenging foreign DNA. We have found that the pVLCNhGH construct, of retrotransposon origin, causes a massive embryo lethality and yet increases the yield of genetic transformation among born animals of both species compared to viral constructs. Arrest of embryonic development is a DNA dose‐dependent effect, which is observed with high DNA doses, while lower doses are compatible with development. Finally, the overall efficiency of sperm‐mediated gene transfer is higher when ejaculated, versus epididymal, spermatozoa are used. We suggest that this difference is related to the highly efficient apoptotic response in epididymal compared to ejaculated spermatozoa, triggered by the interaction of exogenous DNA molecules with the sperm membrane. Mol. Reprod. Dev. 56:301–305, 2000.

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. Mol. Reprod. Dev. 79:118–127, 2012.

Normal and cancer-prone human cells respond differently to extremely low frequency magnetic fields

Human lymphoblastoid cells of normal origin and from genetic instability syndromes, i.e. Fanconi anemia (FA) group C and ataxia telangectasia, were continuously exposed to extremely low frequency magnetic field (ELF‐MF). We report that ELF‐MF, though not perturbing cell cycle progression, increases the rate of cell death in normal cell lines. In contrast, cell death is not affected in cells from genetic instability syndromes; this reflects a specific failure of the apoptotic response. Reintroduction of complementation group C in FA cells re‐established the apoptotic response to ELF‐MF. Thus, genes implicated in genetic instability syndromes are relevant in modulating the response of cells to ELF‐MF.

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.

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.