miR-205 regulates basement membrane deposition in human prostate: implications for cancer development.

The basement membrane (BM) is a layer of specialized extracellular matrix that surrounds normal prostate glands and preserves tissue integrity. Lack or discontinuity of the BM is a prerequisite for tumor cell invasion into interstitial spaces, thus favoring metastasis. Therefore, BM maintenance represents a barrier against cancer development and progression. In the study, we show that miR-205 participates in a network involving ΔNp63α, which is essential for maintenance of the BM in prostate epithelium. At the molecular level, ΔNp63α is able to enhance miR-205 transcription by binding to its promoter, whereas the microRNA can post-transcriptionally limit the amount of ΔNp63α protein, mostly by affecting ΔNp63α proteasomal degradation rather than through a canonical miRNA/target interaction. Functionally, miR-205 is able to control the deposition of laminin-332 and its receptor integrin-ß4. Hence, pathological loss of miR-205, as widely observed in prostate cancer, may favor tumorigenesis by creating discontinuities in the BM. Here we demonstrate that therapeutic replacement of miR-205 in prostate cancer (PCa) cells can restore BM deposition and 3D organization into normal-like acinar structures, thus hampering cancer progression.

Apollon gene silencing induces apoptosis in breast cancer cells through p53 stabilisation and caspase-3 activation.

We analysed the effects of small interfering RNA (siRNA)-mediated silencing of Apollon, a member of the inhibitors of apoptosis protein family, on the proliferative potential and ability of human breast cancer cell lines to undergo apoptosis. In wild-type p53 ZR75.1 cells, Apollon knockdown resulted in a marked, time-dependent decline of cell growth and an increased rate of apoptosis, which was associated with p53 stabilisation and activation of the mitochondrial-dependent apoptotic pathway. Pre-incubation of cells with a p53-specific siRNA resulted in a partial rescue of cell growth inhibition, as well as in a marked reduction of the apoptotic response, indicating p53 as a major player in cell growth impairment consequent on Apollon silencing. Apollon knockdown induced consistently less pronounced anti-proliferative and pro-apoptotic effects in mutant p53 MDA-MB-231 cells than in ZR75.1 cells. Furthermore, the activation of caspase-3 seemed to be essential for the induction of apoptosis after Apollon knockdown, as the Apollon-specific siRNA had no effect on the viability of caspase-3-deficient, wild-type p53 MCF-7 cells or the ZR75.1 cells after RNA interference-mediated caspase-3 silencing. Our results indicate that p53 stabilisation and caspase-3 activation concur to determine the apoptotic response mediated by Apollon knockdown in breast cancer cells, and suggest Apollon to be a potential new therapeutic target for this malignancy.

Photochemically enhanced delivery of a cell-penetrating peptide nucleic acid conjugate targeting human telomerase reverse transcriptase: effects on telomere status and proliferative potential of human prostate cancer cells.

OBJECTIVES: Peptide nucleic acids (PNAs) are DNA mimics that have been demonstrated to be efficient antisense/antigene tools in cell-free systems. However, their potential as in vivo regulators of gene expression has been hampered by their poor uptake by living cells, and strategies need to be developed for their intracellular delivery. This study has aimed to demonstrate the possibility (i) of efficiently delivering a PNA, which targets mRNA of the catalytic component of human telomerase reverse transcriptase (hTERT), into DU145 prostate cancer cells through a combined approach based on conjugation of the PNA to Tat internalizing peptide (hTERT-PNA-Tat) and subsequent photochemical internalization, and (ii) to interfere with telomerase function. MATERIALS AND METHODS: Treated cells were analysed for telomerase activity, hTERT expression, growth rate, ability to undergo apoptosis and telomere status. RESULTS: After exposure to light, DU145 cells treated with hTERT-PNA-Tat and the photosensitiser TPPS2a showed dose-dependent inhibition of telomerase activity, which was accompanied by marked reduction of hTERT protein expression. A dose-dependent decline in DU145 cell population growth and induction of caspase-dependent apoptosis were also observed from 48 h after treatment. Such an antiproliferative effect was associated with the presence of telomeric dysfunction, as revealed by cytogenetic analysis, in the absence of telomere shrinkage, and with induction of DNA damage response as suggested by the increased expression of gamma-H2AX. CONCLUSIONS: Our results (i) indicate photochemical internalization as an efficient approach for intracellular delivery of chimaeric PNAs, and (ii) corroborate earlier evidence suggesting pro-survival and anti-apoptotic roles of hTERT, which are independent of its ability to maintain telomere length.

Targeting telomerase by antisense-based approaches: perspectives for new anti-cancer therapies.

Oligonucleotide-based therapies have been under investigation for many years, and different antisense oligomers are being tested in clinical trials on patients with cancer and other diseases. Since telomerase reactivation has been defined as one of the six hallmarks of cancer because of the enzyme's ability to provide tumor cells with unlimited proliferative potential, antisense-based approaches, aimed to inhibit the core enzyme components, could represent innovative anticancer therapies. Overall, available information indicates antisense-based strategies as powerful tools to inhibit telomerase and interfere with tumor cell proliferative potential. Specifically, cancer cell growth arrest was observed in several tumor models as a consequence of telomere shortening in the presence of prolonged telomerase inhibition. However, in other studies, antisense-based treatments caused rapid loss of tumor cell viability and induced apoptosis independently of telomere attrition. The results would suggest that telomerase inhibition affects tumor cell growth by mechanisms that are dependent as well as independent of the enzyme telomere elongating activity. However, the role of telomerase in tumorigenesis and tumor progression, beyond the classical mechanism of telomere lengthening, needs to be further investigated to provide a better rationale for the design and development of antitelomerase-based therapies in clinical oncology.

Oligomer-mediated modulation of hTERT alternative splicing induces telomerase inhibition and cell growth decline in human prostate cancer cells.

The expression of telomerase in human cells is strictly controlled by multiple mechanisms including transcription and alternative splicing of telomerase reverse transcriptase (hTERT). In this study, we demonstrated the possibility of modulating the hTERT splicing pattern in DU145 human prostate carcinoma cells through the use of 2'-O-methyl-RNA phosphorothioate oligonucleotides targeting the splicing site located between intron 5 and exon 6 in the hTERT pre-mRNA. An 18-h oligonucleotide exposure induced a decrease in the full-length hTERT transcript and a concomitant increase in the alternatively spliced transcripts, which resulted in significant inhibition of telomerase catalytic activity. Moreover, exposure to the R7 oligomer (which induced the most pronounced modulation of the hTERT splicing pattern and the greatest telomerase inhibition) caused a marked reduction in DU145 cell growth and the induction of apoptosis starting 2 days after treatment. Such data support the concept that down-regulation of hTERT expression can cause short-term effects on tumour cell growth, which are telomere-shortening independent.

Approaches for the inhibition of human telomerase based on the use of peptide nucleic acids and hammerhead ribozymes.

The ability of peptide nucleic acids and hammerhead ribozymes, which target different subunits of human telomerase, to efficiently inhibit the enzyme's catalytic activity has been clearly demonstrated in several in vitro studies carried out in human immortalized and cancer cell lines. However, the actual efficacy of these molecules still needs to be validated in in vivo human tumor models, and such validation appears to be largely dependent on the development of reliable systems for their intracellular delivery.

Targeting human telomerase by antisense oligonucleotides and ribozymes.

Human telomerase is a ribonucleoprotein enzyme complex that enables cells to maintain telomere length, allowing indefinite replicative capacity. The notion that telomerase is reactivated in 80-90% of human cancers has led to the proposal of telomerase as a promising therapeutic target for novel anticancer interventions. Due to its inherent accessibility to nucleic acids, telomerase appears an ideal target for strategies based on the use of antisense oligonucleotides and ribozymes that target its RNA template. In this review a summary of the different antisense- and ribozyme-based approaches used thus far to inhibit telomerase activity in human cancer cells is provided. All these strategies significantly inhibited the enzyme's catalytic activity in in vitro and in vivo tumor models. However, while in some studies tumor cell growth arrest was observed as a consequence of telomere shortening after prolonged telomerase inhibition, other studies have shown that antisense- and ribozyme-based treatments targeting telomerase induced rapid loss (i.e. within a few days) of tumor cell viability with concomitant apoptosis. In the latter case it is unlikely that cell death was related to telomere erosion since the cells would not have undergone enough divisions to significantly shorten their telomeres. A possible explanation is that telomerase inhibitors may induce apoptosis in cancer cells directly by interfering with the capping function of the enzyme. Overall, the available results indicate antisense oligonucleotides and ribozymes as good tools to inhibit telomerase and suggest that abrogation of telomerase activity may affect tumor cell proliferation also through pathways that are not dependent on telomere erosion.

Possible regulation of telomerase activity by transcription and alternative splicing of telomerase reverse transcriptase in human melanoma.

To investigate the regulatory mechanisms of telomerase activity in human melanoma cells, we assessed the enzyme's catalytic activity and the expression of the telomerase subunits, the human telomerase RNA, the human telomerase-associated protein, and the human telomerase reverse transcriptase, in 52 melanoma lesions. Eight normal skin specimens were also studied. Telomerase activity was detected in 84.6% of melanomas, whereas all skin specimens were telomerase negative. Human telomerase-associated protein mRNA and human telomerase RNA were constitutively expressed in all melanoma and skin specimens. Although at a variable level of expression, human telomerase reverse transcriptase mRNA was detected in all but one melanomas, whereas it was never present in skin samples. Reverse transcriptase-polymerase chain reaction experiments were performed using primers within the reverse transcriptase domain of human telomerase reverse transcriptase and revealed the presence of multiple alternatively spliced transcripts in melanoma specimens. Among the 44 telomerase-positive melanomas, one showed the full-length transcript alone whereas in all other specimens a full-length message was present with different combinations of alternatively spliced variants. In these tumors the expression of the full-length transcript was generally equal to or higher than that of the alternatively spliced variants. The ratio full-length transcript to alternatively spliced species ranged from 0.6 to 5.26, with a median value of 1.18. Among the seven telomerase-negative melanomas, one displayed the beta deletion transcript alone, whereas in the remaining six tumors weak expression of the full-length transcript and a more abundant level of alternatively spliced transcripts were found. In these cases human telomerase reverse transcriptase ratio ranged from 0.09 to 1.1, with a median value of 0.40. The results suggest that transcription and alternative splicing of human telomerase reverse transcriptase are regulatory mechanisms controlling telomerase activity in melanoma.

Attenuation of telomerase activity does not increase sensitivity of human melanoma cells to anticancer agents.

In tumour cells, replicative immortality is attained through stabilisation of telomeres by telomerase. Recent evidence suggests that telomerase plays an anti-apoptotic role. Since apoptosis is the primary mode of cell death induced by several drugs, telomerase could be involved in determining the chemosensitivity profile of tumour cells. We investigated whether inhibition of telomerase activity through a hammerhead ribozyme targeting the RNA template of telomerase influences the susceptibility of human melanoma cells to a variety of anticancer agents (platinum compounds, taxanes, topoisomerase I inhibitors). The ribozyme sequence was inserted into an expression vector and the JR8 human melanoma cell line was transfected with it. The cell clones obtained showed a reduced telomerase activity. Growth inhibition curves generated after exposure of ribozyme-transfectant clones to individual drugs were superimposable to those obtained from parental cells. Moreover, telomerase inhibition did not promote apoptosis as a cellular response to drug treatment. Overall, our results indicate that downregulation of telomerase activity does not increase the sensitivity of melanoma cells to anticancer drugs.

Inhibition of telomerase activity by a cell-penetrating peptide nucleic acid construct in human melanoma cells.

We investigated the effect of two peptide nucleic acids (PNAs), which are complementary to the RNA component of human telomerase, on the catalytic activity of the enzyme. PNAs induced a dose-dependent reduction of telomerase activity in cell extracts from human melanoma cell lines and surgical specimens. To down-regulate telomerase in intact cells, we generated a chimeric molecule synthesized by coupling the 13-mer PNA to the Antennapedia peptide. The PNA construct induced a dose- and time-dependent inhibition of telomerase activity. However, a 20-day exposure to the PNA construct only caused a slight increase in melanoma cell doubling time and failed to induce any telomere shortening.

Telomerase activity and telomere length in human ovarian cancer and melanoma cell lines: correlation with sensitivity to DNA damaging agents.

Since telomerase plays a role in cellular resistance to apoptosis, which is the primary mode of cell death induced by several drugs, telomerase could be involved in determining the chemosensitivity profile of tumor cells. Thus, we investigated the relationship between telomerase activity, telomere length and chemosensitivity to effective antitumor agents in a panel of human melanoma and ovarian cancer cell lines. Telomerase activity, as detected by the telomeric repeat amplification protocol, ranged from 0.58 to 1.10 arbitrary units in individual cell lines, with similar median values for melanoma and ovarian carcinoma cell lines (0.80 vs. 0.90). Telomeres were generally longer in melanoma than in ovarian carcinoma cell lines, with a more than 2-fold median telomere restriction fragment length (7.74 vs. 3.82 kb). No significant correlation was evidenced between the two telomere-related parameters and cell population doubling time, DNA index or TP53 gene status. No precise relation was found between telomerase activity and cellular sensitivity to different DNA damaging agents including doxorubicin, cisplatin and the multinuclear platinum compound BBR 3464. In contrast, longer telomeres were associated to resistance to the drugs, even though the association reached statistical significance only for cisplatin. Since platinum compounds may have affinity for telomere sequences, it is conceivable that the interaction is relevant for drug sensitivity/resistance status depending on telomere length.

Inhibition of telomerase activity by a hammerhead ribozyme targeting the RNA component of telomerase in human melanoma cells.

Reactivation of telomerase, an RNA-dependent DNA polymerase that synthesizes new telomeric repeats at the end of chromosomes, is a very common feature in human cancers. Telomerase is thought to be essential in maintaining the proliferative capacity of tumor cells and, as a consequence, it could represent an attractive target for new anti-cancer therapies. In this study, we generated a hammerhead ribozyme composed of a catalytic domain with flanking sequences complementary to the RNA component of human telomerase and designed to cleave specifically a site located at the end of the telomerase template sequence. In vitro the ribozyme induced cleavage of a synthetic RNA substrate obtained by cloning a portion of the RNA component of human telomerase. The extent of cleavage was dependent on the ribozyme/substrate ratio as well as the Mg2+ concentration. Moreover, when added to cell extracts from two human melanoma cell lines (JR8 and M14), or three melanoma surgical specimens, the ribozyme inhibited telomerase activity in a concentration- and time-dependent manner. When the ribozyme was delivered to growing JR8 melanoma cells by (N-(1-(2,3 dioleoxyloxy)propil)-N,N,N trimethylammonium methylsulfate-mediated transfer, a marked inhibition of telomerase activity was observed. Next, the ribozyme sequence was cloned in an expression vector and JR8 cells were transfected with it. The cell clones obtained showed a reduced telomerase activity and telomerase RNA levels and expressed the ribozyme. Moreover, ribozyme transfectants had significantly longer doubling times than control cells and showed a dendritic appearance in monolayer culture. No telomere shortening, however, was observed in these clones. Overall, our results indicate that the hammerhead ribozyme is a potentially useful tool for the inactivation of telomerase in human tumors.

Macrophage populations of different origins have distinct susceptibilities to lipid peroxidation induced by beta-haematin (malaria pigment).

We investigated the susceptibility of peritoneal mouse macrophages and macrophage and microglia cell lines to the peroxidative activity of beta-haematin, the synthetic polymer identical to native malaria pigment. The extent of lipid peroxidation, measured as production of thiobarbituric acid reactive substances (TBARS), was greater for peritoneal macrophages than for cell lines and microglia cells. TBARS production apparently was not attributable to the release of free iron from the protoporphyrin moiety, but related to lower glutathione content and different lipid composition of the cell membrane. These findings offer a new interpretation for the contentious immunomodulatory effects of beta-haematin reported for phagocytes of different origins.

Schedule-dependent modulation of idarubicin cytotoxicity by lonidamine in human lymphoma cell lines.

The ability of lonidamine (LND), an energolytic derivative of indazol-carboxylic acid, to modulate the cytotoxic activity of idarubicin (IDA) and doxorubicin (DX) was investigated in two human lymphoma cell lines (H9 and U937). A different pattern of interaction between the drugs was observed as a function of treatment sequence. Specifically, a 24-h postincubation with a non-cytotoxic concentration of LND (75 mu M) increased the activity of a 1-h anthracycline treatment in both cell lines. However, the extent of potentiation for IDA was more than twofold that of DX. No enhancement of anthracycline activity was observed when LND preceded IDA. For comparative purposes, the modulating effect of all-trans-retinoic acid (ATRA) on the cytotoxicity of IDA was evaluated according to different treatment schemes in both lymphoma cell lines. In U937 cells, which undergo monocytic differentiation after exposure to retinoids, a marked increase in LDA activity was obtained following a 48-h postincubation with 1.5 mu M ATRA. No potentiation of anthracycline activity was obtained using the opposite drug sequence. In H9 cells, no significant interference between ATRA and IDA was observed independent of the modality of drug administration. The ability of LND to potentiate IDA activity, and the consideration that LND causes side effects different from those caused by anthracyclines, make this compound an attractive candidate for multidrug combination therapy in hematological neoplasms.

Overexpression of protein kinase C epsilon is oncogenic in rat colonic epithelial cells.

We have analysed the expression of five protein kinase C [PKC] isoforms in an in vitro model using nontumorigenic rat colonic epithelial cells FRC/TEX CL D [D/WT] and in the related tumorigenic Ha-ras-transformed FRC/TEX CL D/H-ras line [D/ras]. The PKC subspecies alpha, delta, epsilon and xi were expressed at the protein level in both D/WT and D/ras cells, while beta PKC was undetectable in both lines. The levels of expression of the delta and xi isoforms were similar in D/WT and D/ras cells. Alpha PKC expression was decreased and epsilon PKC was increased in D/ras cells compared to the D/WT line. To assess whether overexpression of epsilon PKC was linked to the transformed phenotype, we have generated from D/WT cells two clones (D/epsilon-5 and D/epsilon-9) which stably overexpress epsilon PKC about fivefold. Overexpression of epsilon PKC caused marked morphological changes in both transfected clones, which were accompanied by increased saturation densities and anchorage-independent colony formation in semisolid agar. These growth effects were attenuated or reversed by chronic incubation with phorbol 12-myristate 13-acetate. Furthermore, D/epsilon-5 and D/epsilon-9 cells formed tumors in athymic nude mice with 100% incidence while the parental D/WT or vector alone (D/MV12) controls produced no tumors. We conclude that epsilon PKC can act as an oncoprotein when modestly overproduced in nontumorigenic D/WT colonic cells, and that this isoform of PKC may be linked to ras-modulated signal transduction leading to neoplastic transformation in colonic epithelium.