The Oncogenic Signaling Pathways in BRAF-Mutant Melanoma Cells are Modulated by Naphthalene Diimide-Like G-Quadruplex Ligands.

Melanoma is the most aggressive and deadly type of skin cancer. Despite the advent of targeted therapies directed against specific oncogene mutations, melanoma remains a tumor that is very difficult to treat, and ultimately remains incurable. In the past two decades, stabilization of the non-canonical nucleic acid G-quadruplex structures within oncogene promoters has stood out as a promising approach to interfere with oncogenic signaling pathways in cancer cells, paving the way toward the development of G-quadruplex ligands as antitumor drugs. Here, we present the synthesis and screening of a library of differently functionalized core-extended naphthalene diimides for their activity against the BRAFV600E-mutant melanoma cell line. The most promising compound was able to stabilize G-quadruplexes that formed in the promoter regions of two target genes relevant to melanoma, KIT and BCL-2. This activity led to the suppression of protein expression and thus to interference with oncogenic signaling pathways involved in BRAF-mutant melanoma cell survival, apoptosis, and resistance to drugs. This G-quadruplex ligand thus represents a suitable candidate for the development of melanoma treatment options based on a new mechanism of action and could reveal particular significance in the context of resistance to targeted therapies of BRAF-mutant melanoma cells.

Down-Regulation of the Androgen Receptor by G-Quadruplex Ligands Sensitizes Castration-Resistant Prostate Cancer Cells to Enzalutamide.

Stabilization of the G-quadruplexes (G4s) within the androgen receptor (AR) gene promoter to block transcription may represent an innovative approach to interfere with aberrant AR signaling in castration resistant prostate cancer (CRPC). A library of differently functionalized naphthalene diimides (NDIs) was screened for their ability to stabilize AR G4s: the core-extended NDI (7) stood out as the most promising ligand. AR-positive cells were remarkably sensitive to 7 in comparison to AR-negative CRCP or normal prostate epithelial cells; 7 induced remarkable impairment of AR mRNA and protein amounts and significant perturbations in the expression levels of KLK3 and of genes involved in the activation of AR program via feedback mechanisms. Moreover, 7 synergistically interacted with Enzalutamide, an inhibitor of AR signaling used in second-line therapies. Overall, our data show that stabilization of AR G4s may represent an alternative treatment options for CRPC and other malignancies relying on aberrant androgen signaling.

miR-380-5p-mediated repression of TEP1 and TSPYL5 interferes with telomerase activity and favours the emergence of an "ALT-like" phenotype in diffuse malignant peritoneal mesothelioma cells.

BACKGROUND: Understanding the molecular/cellular underpinnings of diffuse malignant peritoneal mesothelioma (DMPM), a fatal malignancy with limited therapeutic options, is of utmost importance for the fruitful management of the disease. In this context, we previously found that telomerase activity (TA), which accounts for the limitless proliferative potential of cancer cells, is prognostic for disease relapse and cancer-related death in DMPM patients. Consequently, the identification of factors involved in telomerase activation/regulation may pave the way towards the development of novel therapeutic interventions for the disease. Here, the capability of miR-380-5p, a microRNA negligibly expressed in telomerase-positive DMPM clinical specimens, to interfere with telomerase-mediated telomere maintenance and, hence, with cancer cell growth was assessed on preclinical models of DMPM. METHODS: DMPM cells were transfected with a miR-380-5p synthetic precursor, and the effects of miRNA replacement were evaluated in terms of growing capability, induction of apoptosis and interference with TA. Reiterated weekly transfections were also performed in order to analyse the phenotype arising upon prolonged miR-380-5p reconstitution in DMPM cells. RESULTS: The ectopic expression of miR-380-5p elicited a remarkable inhibition of TA and resulted in DMPM cell growth impairment and apoptosis induction. In particular, we demonstrated for the first time that these effects were the result of a molecular circuitry converging on telomerase associated protein 1 (TEP1), where the miRNA was able to target the gene both directly in unconventional targeting modality and indirectly via p53 accumulation consequent to miRNA-mediated downregulation of testis-specific protein, Y-encoded-like 5 gene. Moreover, miR-380-5p did not cause telomere attrition and cell growth arrest in long-term DMPM transfectants, which in turn showed slightly elongated telomeres and molecular features (e.g. c-circle DNA and reduced expression levels of chromatin remodeler ATRX) resembling an alternative lengthening of telomeres (ALT) phenotype. CONCLUSIONS: miR-380-5p interferes with TA in DMPM cells by targeting TEP1. Notably, in the long-term setting, miR-380-5p-mediated impairment of TA did not result in telomere attrition. Instead, a phenotype reminiscent of ALT emerged in DMPM cells as possible compensatory pathway that safeguards DMPM cell growth, an event that may be regarded as a potential resistance mechanism to anticancer therapies based on telomerase inhibitors.

CPAM type 2-derived mesenchymal stem cells: Malignancy risk study in a 14-month-old boy.

INTRODUCTION: The association between congenital pulmonary airway malformations (CPAM) and malignancy is reported in the literature. Interactions between the tumor, immune, and mesenchymal stromal/stem cells (MSCs) have been recognized as crucial for understanding tumorigenesis. We characterized MSCs isolated from CPAM lesions in order to define potential malignancy risks. METHODS: CPAM II pulmonary tissue was used for MSC expansion; a "healthy" lung section from the same child was used as a comparator. Morphology, immunophenotype, differentiation and immunological capacity, proliferative growth, gene signature telomerase activity, and in vivo tumorigenicity in nude mice were evaluated. RESULTS: MSCs were successfully isolated and propagated from CPAM tissue. CPAM-MSCs presented the typical MSC morphology and phenotype, while exhibiting high proliferative capacity, reaching confluence at a median time of 5 days as well as differentiation capabilities. CPAM-MSCs at early passages were not neoplastic and chromosomally normal, even though unbalanced chromosomal rearrangements were noted by molecular karyotype. CONCLUSIONS: CPAM-MSCs exhibited specific features similar to tumor derived MSCs. Whilst there was no evidence of malignant transformation in the cystic tissue, our results provide evidence that this abnormal tissue has malignant potential. MSCs are considered important players in the tumor microenvironment and they have been closely linked to regulation of tumor survival, growth, and progression. Thus, early lesion resection also in asymptomatic patients might be indicated to exclude that the microenvironment may be potentially permissive to cancer development.

Strategies to Strike Survival Networks in Cancer.

The machinery that maintains cellular and tissue homeostasis in a healthy individual is recruited and hijacked by cancer cells to support tumor growth and progression. Activation of often unpredictable alternative or complementary signaling pathways allows cancer cells to bypass the intrinsic self-destructive machinery and the limited replicative potential present in every cell for correct homeostasis maintenance. Therefore, evasion/resistance to apoptosis/cell death, self-sufficiency in growth/survival signals, and limitless replicative potential remain undoubted hallmarks of cancer, contributing to drug resistance. Herein, we specifically review antitumor strategies involving agents targeting deregulated receptor tyrosine kinases, selected epigenetic modifications, the ubiquitin-proteasome system, the unfolded protein response, the apoptotic pathway, and peculiar nucleic acid structures, with the aim of highlighting the mechanisms underlying favorable drug interaction. In this context, we focus on preclinical studies that have already been translated into clinical investigation. Particular emphasis is devoted to strategies effective in solid tumors because of the peculiarity and distinctive features of solid tumors in terms of drug delivery and relative to the impact of the tumor microenvironment.

Emerging Role of G-quadruplex DNA as Target in Anticancer Therapy.

DNA has represented the most exploited target for the development of anticancer agents. It is now established that DNA may assume a variety of non-B conformations. This evidence has generated a total novel wave of interest in DNA as a cancer-associated target, since its distinct non-B structures may be regarded as sites for selective therapeutic intervention. G-quadruplexes are peculiar non-B DNA conformations that may form within guaninerich nucleic acid sequences. They are generated by a core of two or more vertically stacked G-quartets (i.e., the square planar arrangement of four guanine residues) held together by intervening loops of variable length. The evidence that G-quadruplexes are highly polymorphic and overrepresented within human genome points out at such non-B DNA conformations as druggable sites amenable of targeting by small molecules. In the present paper we will provide a concise overview on the emerging role of G-quadruplex structures forming within telomeres, gene promoters and mitochondrial DNA as a promising therapeutic target in cancer. In this context, a variety of small molecules has been documented to have excellent G-quadruplex binding/stabilizing properties and to exert good antiproliferative and antitumor activity in several in vitro and in vivo models of human cancers. Pieces of evidence indicate that targeting G-quadruplexes may represent an innovative and fascinating approach for the therapeutic management of the neoplastic disease. However, several issues still need to be addressed both at chemical and biological level before G-quadruplex-interacting molecules will turn out into effective therapeutic agents. Nevertheless, this has been an exciting, though sometime subdued, field of research over the last century. The continued improvements in methodologies and the development of specific tools will contribute not only to achieve the design and development of potentially novel anticancer approaches but also to deepen our knowledge of G-quadruplex biology and, consequently, of cancer at molecular level.

Naphthalene diimides as red fluorescent pH sensors for functional cell imaging.

A small library of hydrosoluble naphthalene diimides (NDIs) was designed and synthesized, as cell permeable pH "turned-on" fluorescent sensors, for cellular applications. The NDIs exhibit a non-emitting twisted intramolecular charge transfer (TICT) state, which has been described by a DFT computational investigation. These NDIs do not emit as a free base, but they become strong emitters when protonated. Switching of the red emission was achieved in the pH window 2.5-6, tuning steric and electronic features of the amine moiety. The least acidic protonated NDI (pKa 5.1), was investigated in normal and cancer cells. Its selective redistribution in cancer cells from acidic vesicular organelles to the cytoplasm and the nucleus describes an effective application of these NDIs as a valuable functional tool.

Assessment of gene promoter G­quadruplex binding and modulation by a naphthalene diimide derivative in tumor cells.

Naphthalene diimide (NDI) derivatives have shown high affinity for telomeric guanine (G)­quadruplexes and good antiproliferative activity in different human tumor experimental models. A trisubstituted compound (H­NDI­NMe2) has been reported to stabilize the telomeric G­quadruplex and to cause telomere dysfunction and downregulation of telomerase expression. We further investigated its mechanism of action by analyzing the capability of the molecule to interfere with the expression levels of oncogenes, such as MYC, telome-rase reverse transcriptase (TERT), KIT and BCL2, known to bear G­quadruplex­forming sequences within their promoters, in human tumor cell lines of different histological origin. Exposure to H­NDI­NMe2 resulted in a cell type­dependent perturbation of the expression levels of the four selected genes. Biophysical and molecular analyses revealed that H­NDI­NMe2 bound with high affinity and effectively stabilized mainly MYC and BCL2, which share long sequences and the possibility of multiple G­quadruplex folding. The mRNA levels of both genes, but not protein amounts were affected by NDI treatment. Global gene expression analysis showed modulation of genes implicated in telomere function and mechanisms of cancer; however, G­quadruplex­mediated regulation of gene expression by H­NDI­NMe2 was largely dependent on the cell context. These data indicate that a deeper knowledge on the molecular mechanisms and biological effects of G­quadruplex structures is still needed to help developing new effective anticancer agents.

MicroRNA-dependent regulation of telomere maintenance mechanisms: a field as much unexplored as potentially promising.

The activation of telomere maintenance mechanisms, which rely on telomerase reactivation or on a recombination-based process known as alternative lengthening of telomeres, guarantees a limitless proliferative potential to human tumor cells. To date, the molecular underpinnings that drive the activation of telomere maintenance mechanisms during tumorigenesis are poorly understood, but there are indications that complex signaling networks might be involved. Since telomerase activity has been mainly detected in tumors of epithelial origin and the alternative lengthening of telomere mechanisms is more frequently expressed in mesenchymal and neuroepithelial cancers, it could be hypothesized that cell-type specific mechanisms can favor their activation during tumor development. In this context, microRNAs - small non coding RNAs that regulate gene expression at post-transcriptional level - have emerged as key players in the development and progression of human cancers, being involved in the control of all the typical features of cancer cells, including the limitless replicative potential. In the present review, we will summarize the recent findings concerning the identification and biological validation of microRNAs which may play a role in the regulation of telomere biology as well as of the mechanisms that govern telomere maintenance.

G-quadruplex structures in the human genome as novel therapeutic targets.

G-quadruplexes are secondary structures that may form within guanine-rich nucleic acid sequences. Telomeres have received much attention in this regard since they can fold into several distinct intramolecular G-quadruplexes, leading to the rational design and development of G-quadruplex­stabilizing molecules. These ligands were shown to selectively exert an antiproliferative and chemosensitizing activity in in vitro and in vivo tumor models, without appreciably affecting normal cells. Such findings point to them as possible drug candidates for clinical applications. Other than in telomeres, G-quadruplexes may form at additional locations in the human genome, including gene promoters and untranslated regions. For instance, stabilization of G-quadruplex structures within the promoter of MYC, KIT, or KRAS resulted in the down-regulation of the corresponding oncogene either in gene reporter assays or in selected experimental models. In addition, the alternative splicing of a number of genes may be affected for a therapeutic benefit through the stabilization of G-quadruplexes located within pre-mRNAs. It is now emerging that G-quadruplex structures may act as key regulators of several biological processes. Consequently, they are considered as attractive targets for broad-spectrum anticancer therapies, and much effort is being made to develop a variety of ligands with improved G-quadruplex recognition properties. Quarfloxin, a fluoroquinolone derivative designed to target a G-quadruplex within ribosomal DNA and disrupt protein-DNA interactions, has entered clinical trials for different malignancies. This review will provide some hints on the role of G-quadruplex structures in biological processes and will evaluate their implications as novel therapeutic targets.

Tel1 and Rad51 are involved in the maintenance of telomeres with capping deficiency.

Vertebrate-like T2AG3 telomeres in tlc1-h yeast consist of short double-stranded regions and long single-stranded overhang (G-tails) and, although based on Tbf1-capping activity, they are capping deficient. Consistent with this idea, we observe Y' amplification because of homologous recombination, even in the presence of an active telomerase. In these cells, Y' amplification occurs by different pathways: in Tel1(+) tlc1h cells, it is Rad51-dependent, whereas in the absence of Tel1, it depends on Rad50. Generation of telomeric G-tail, which is cell cycle regulated, depends on the MRX (Mre11-Rad50-Xrs2) complex in tlc1h cells or is MRX-independent in tlc1h tel1Δ mutants. Unexpectedly, we observe telomere elongation in tlc1h lacking Rad51 that seems to act as a telomerase competitor for binding to telomeric G-tails. Overall, our results show that Tel1 and Rad51 have multiple roles in the maintenance of vertebrate-like telomeres in yeast, supporting the idea that they may participate to evolutionary conserved telomere protection mechanism/s acting at uncapped telomeres.

Autophagy acts as a safeguard mechanism against G-quadruplex ligand-mediated DNA damage.

G-quadruplex ligands have attracted considerable interest as novel anticancer therapeutics due to their capability to interfere with guanosine-rich DNA/RNA sequences, such as telomeres. Elucidation of the structures of telomeric G-quadruplexes has led, in the past few years, to the rational development of effective G-quadruplex-stabilizing small molecules. In the present study, we showed that short-term exposure of melanoma cells to Ant1,5--an anthracene-based ligand able to stabilize telomeric G-quadruplexes--impaired cell growth without inducing cell senescence or apoptosis. Conversely, drug-treated cells were characterized by the occurrence of typical biochemical and morphological features associated with autophagy, such as an increase in the lipidated form of the autophagic marker LC3B and the accumulation of autophagosomes. Such drug-induced autophagy occurred as a consequence of DNA damage induction, at least in part dependent on drug-mediated telomere uncapping, through a pathway converging on the cyclin-dependent kinase inhibitor 1A (CDKN1A/p21). Indeed, melanoma cells depleted for CDKN1A did not show evidence of autophagic markers upon exposure to Ant1,5. The inhibition of autophagy by a pharmacologic inhibitor or through RNAi-mediated depletion of the ATG5 gene enhanced the cytotoxic activity of Ant1,5, as revealed by the marked increase in drug-induced apoptosis. Our data outline a molecular scenario in which G-quadruplex ligand-induced telomeric dysfunctions and DNA damage are translated into an autophagic response and provide the first evidence of autophagy as a safeguard mechanism activated by melanoma cells to counteract G-quadruplex ligand-mediated cellular stress.

Telomere maintenance mechanisms in malignant peripheral nerve sheath tumors: expression and prognostic relevance.

This study investigated the prevalence and the prognostic relevance of the 2 known telomere maintenance mechanisms (TMMs), telomerase activity (TA) and alternative lengthening of telomeres (ALT), in malignant peripheral nerve sheath tumors (MPNST). In 57 specimens from 49 patients with MPNST (35 sporadic, 14 neurofibromatosis type 1-related), TA was determined using the telomeric repeat amplification protocol, and ALT was detected by assaying ALT-associated promyelocytic leukemia bodies (APB) and terminal restriction fragment (TRF) length distribution. TA or ALT (defined on the basis of APB) alone was found in 24.6% or 26.3% of the lesions, respectively, whereas 6 cases (10.5%) were TA+/ALT+. A concordance between APB and TRF results in defining the ALT status was observed in 44 of 57 cases (77.2%; P < .0001). TA was more frequently expressed in samples from patients with neurofibromatosis type 1 than in those with sporadic disease (60% vs 29.4%, P = 0.087). In the overall series, TA proved to be prognostic for 5-year disease-specific death (hazard ratio, 3.78; 95% confidence interval [CI], 1.60-8.95; P = .002), even when adjusted for the presence of neurofibromatosis type 1 (hazard ratio, 4.22; 95% CI, 1.804-9.874; P = .001) and margin status after surgery (hazard ratio, 5.78; 95% CI, 2.19-15.26; P < .001). Conversely, ALT did not significantly affect clinical outcome of MPNST using either APB expression (hazard ratio, 1.25; 95% CI 0.54-2.89; P = 0.605) or TRF distribution (hazard ratio, 0.57; 95% CI, 0.17-1.96; P = .375) as the detection approach. Our results indicate for the first time that both TMMs, TA and ALT, are present in MPNST and differentially affect patient prognosis.

Telomeres as targets for anticancer therapies.

INTRODUCTION: The limitless replicative potential of cancer cells relies on telomere integrity (which is guaranteed by a complex interaction between several specialized proteins and telomeric DNA) and the activation of specific mechanisms for telomere length maintenance. Two mechanisms are currently known in human cancer, namely telomerase activity and the alternative lengthening of telomere pathway. EXPERT OPINION: In this review, we summarize the available data concerning the therapeutic strategies proposed thus far and the current challenges posed for the development of innovative telomere-based therapeutic approaches with broad-spectrum anticancer activity and for their translation into the clinical setting. AREAS COVERED: Due to their essential role in tumor cell proliferation, telomere maintenance mechanisms have become extremely attractive targets for the development of new anticancer interventions. Although numerous efforts have been made to identify specific approaches to interfere with telomere maintenance mechanisms in human cancers, the only molecule currently tested in clinical trials is the oligonucleotide GRN163L. However, a growing body of evidence suggests that interfering with telomeres, through the direct targeting of telomeric G-quadruplex structures, may be a valuable antitumor therapeutic strategy, independent of the specific telomere maintenance mechanism operating in the tumor.

Erosion of telomeric 3'-overhangs in white blood cells of aged subjects with high frequency of very short telomeres.

After extended proliferation, cells enter a state of replicative quiescence that is probably due to progressive telomere shortening. It is supposed that changes in telomere structure eventually expose the chromosome ends to undesired recombination events and thus promote cell senescence. The telomeric 3'-overhang is crucial for efficient chromosome capping, but its specific role in telomere shortening and in triggering the senescence program is uncertain. We have addressed this issue by measuring the 3'-overhangs of a human tissue cells aging in vivo. The 3'-overhangs were analyzed in blood samples from 41 individuals aged 91-106 years and 89 individuals ranging from 6 months to 85 years. We found that the overall 3'-overhang length did not significantly change with age, but did, however, find extensively eroded 3'-overhangs in 3 subjects of the 91-106 years cohort and one 61 years old subject affected with Down syndrome. These subjects had 3'-overhang length distributions skewed towards shorter tails, the shortest overall telomere lengths and the highest frequencies of very short telomeres. These data raise the possibility that during ageing very short telomeres with very poor 3'-overhangs can reach a critical point for functional telomeres.

Combined in utero and juvenile exposure of mice to arsenate and atrazine in drinking water modulates gene expression and clonogenicity of myeloid progenitors.

The effects of arsenate (As) and atrazine (Atr) on myeloid progenitors (colony-forming unit-granulocyte/macrophage, CFU-GM) cells derived from bone marrow were studied in male and female mice after combined in utero and juvenile exposure. Female adult mice were treated with arsenate in drinking water during gestation. Then, separate groups of males and females' offspring were exposed for 4 months to atrazine, to additional arsenate or to co-exposure of atrazine and arsenate together in drinking water. In male mice, arsenate and the combined exposure did not modulate the percentage of CFU-GM progenitors, whereas atrazine significantly decreases the clonogenicity of myeloid cells. In females, the percentage of CFU-GM significantly decreased after atrazine exposure did not change with arsenate treatment, but dramatically increased after the combined exposure. The expression of estrogen receptors alpha (ERalpha) and beta (ERbeta) in bone marrow cells was investigated, and an up-regulation of receptor beta was observed in both genders. A gene expression profile was generated using nylon membranes spotted with 1185 cancer-related genes. Results from microarrays indicate that atrazine alone did not stimulate the expression of any of the genes analysed in both male and female. Arsenic induced gene expression modulation only in female. Major significant changes on the gene expression resulted following the co-exposure to arsenic and atrazine in both male and female.

Tumor cells fail to trans-induce telomerase in human umbilical vein endothelial cell cultures.

The shortening of the telomeres that occurs in most somatic cells and untransformed cell cultures is considered a hallmark of cellular senescence. Re-activation of telomerase, which is usually present in immortal cells, avoids telomere shortening and considerably extends the culture life span. Normal human endothelial cells are characterized by an accelerated rate of telomere shortening and reach replicative senescence after a limited number of cell divisions. It has recently been reported that human telomerase reverse transcriptase expression may be strongly up-regulated in human endothelial cells cocultivated with tumor cells. Due to the important implications of this finding on tumor progression, we have extensively analyzed for the presence of telomerase in primary human endothelial cells either cocultivated with tumor cells or grown with tumor-conditioned medium. We found modest, but readily detectable, amounts of telomerase in all human endothelial cell cultures analyzed that disappeared as the cultures approached senescence. Quantitative reverse transcription-PCR also showed a direct correlation between human telomerase reverse transcriptase expression and the proliferative index of the cultures. Nevertheless, we did not find any evidence of induction of telomerase activity by tumor cells in any of the tested conditions. All data indicate that telomerase in human endothelial cells follows an activation program that is strictly associated to the culture growth rate.

Chordoma of the skull base: predictors of tumor recurrence.

OBJECT: Chordomas of the skull base are generally regarded as slow-growing tumors; however, approximately 20% of these lesions have been shown to recur as early as 1 year postsurgery. The classic pathological paradigms are poor predictors of outcome, and additional markers are needed to identify patients at risk for early tumor recurrence. In this study the authors describe such a marker. METHODS: In a series of 26 patients with chordomas of the skull base, the authors investigated the relationship between the biological behavior of the tumor, which was determined according to the interval for its recurrence and volume doubling time, and several pathological and molecular features, which included the histological variant, proliferative activity, mutation of p53 protein, expression of human telomerase reverse transcriptase (hTERT) messenger (m)RNA, loss of heterozygosity (LOH), and microsatellite instability. The major finding in this study was that hTERT mRNA expression in chordoma cells identifies those tumors that exhibit unusually fast rates of growth. The expression of hTERT mRNA was frequently associated with mutation of p53 protein, indicating that telomerase dysfunction combines with abnormal p53 function to initiate the unrestrained clonal expansion of the tumor cells. In cases in which the tumor was partially removed, mutation of p53 protein and expression of hTERT mRNA predicted increased doubling time for residual tumor as well as the probability of tumor recurrence. Cell proliferation, as investigated using the Ki-67 method, was significantly related to the tumor doubling time; however, the authors found that the pattern of cell proliferation was not homogeneous throughout the chordoma tissue, and that the proliferative index might change by a factor as high as 8 among different regions of the same tumor. The LOH and microsatellite instability do not seem to affect the prognosis of skull base chordomas. CONCLUSIONS: Reactivation of telomerase in chordomas is a reliable predictor of outcome. The ability to predict the biological behavior of chordomas might have immediate implications in the management of this disease in patients who undergo surgery.

Long telomeric C-rich 5'-tails in human replicating cells.

Telomeres protect the ends of linear chromosomes from abnormal recombination events and buffer them against terminal DNA loss. Models of telomere replication predict that two daughter molecules have one end that is blunt, the product of leading-strand synthesis, and one end with a short G-rich 3'-overhang. However, experimental data from proliferating cells are not completely consistent with this model. For example, telomeres of human chromosomes have long G-rich 3'-overhangs, and the persistence of blunt ends is uncertain. Here we show that the product of leading-strand synthesis is not always blunt but can contain a long C-rich 5'-tail, the incompletely replicated template of the leading strand. We examined the presence of G-rich and C-rich single-strand DNA in fibroblasts and HeLa cells. Although there were no significant changes in the length distribution of the 3'-overhang, the 5'-overhangs were mostly present in S phase. Similar results were obtained using telomerase-negative fibroblasts. The amount and the length distribution of the 5' C-rich tails strongly correlate with the proliferative rate of the cell cultures. Our results suggest that, contrary to what has commonly been supposed, completion of leading-strand synthesis is inefficient and could well drive telomere shortening.

Promotion of regeneration of corticospinal tract axons in rats with recombinant vascular endothelial growth factor alone and combined with adenovirus coding for this factor.

OBJECT: After spinal cord transection in adult rats, the axons of the corticospinal tract (CST) degenerate retrogradely and do not regenerate. This phenomenon is thought to be related to either secondary ischemia or deficiency of growth factors. To overcome the deficiency of both blood flow and growth factors, the authors added exogenous vascular endothelial growth factor (VEGF165) to the transected spinal cord either as recombinant protein alone or combined with an adenovirus coding for VEGF165. Because most growth factors are rapidly inactivated in the extracellular environment, the authors used an adenovirus coding for VEGF165 to maintain its activity for several days. METHODS: In adult rats, the dorsal two thirds of the spinal cord were transected at the T-8 level. In experimental rats, either human recombinant VEGF165 or a combination of this factor and a replication-defective adenovirus coding for VEGF165 (Ad.CMV.VEGF165) was applied at the lesion site. Both recombinant VEGF165 alone and combined with Ad.CMV.VEGF165 were mixed with Matrigel, which is a reconstituted membrane basement protein extract. Control rats received Matrigel alone or Matrigel plus an adenoviral vector containing the LACZ gene (Ad.CMV.LACZ). Thirty days after spinal cord injury, the number of newly formed blood vessels was assessed in the injured area. In addition, the sensorimotor cortex was injected with anterogradely transported horseradish peroxidase (HRP) to label the CST axons in the spinal cord and to evaluate the extent of retrograde axonal degeneration and regeneration. Gene transfer was assessed using semiquantitative reverse transcription-polymerase chain reaction analysis, enzyme-linked immunosorbent assay for human VEGF and beta-galactosidase expression in injured rats treated with Matrigel plus Ad.CMV.LACZ, Matrigel plus Ad.CMV.VEGF165, and untreated injured rats. A strong gene transfer in the spinal cord tissue of adenovirus-treated rats was found from Day 3 to Day 10 postinjury, confirming infection. In the injured spinal cord area, a significant increase of blood vessels (300% over control, p < 0.005) occurred both in rats treated with recombinant VEGF165 alone and in those treated with the combination of recombinant VEGF165 and Ad.CMV.VEGF165. Also, in both of these groups of animals the retrograde degeneration of CST axons was significantly reduced compared with rats treated with Matrigel alone or Matrigel plus Ad.CMV.LACZ. Furthermore, in rats treated with recombinant VEGF165 alone or combined with Ad.CMV.VEGF165, a few HRP-labeled CST axons, which were not detectable in control rats, were seen distal to the spinal cord injury, indicating some regeneration across the injured area. CONCLUSIONS: These results indicate that locally applied VEGF exerts angiogenic as well as neurotrophic effects in the injured spinal cord of rats.