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The human antiapoptotic bcl-2 gene has been discovered in t(14;18) B-cell leukemias/lymphomas because of its overexpression caused at a transcriptional control level by the bcl-2/IgH fusion gene. We were the first to disclose the post-transcriptional control of bcl-2 expression mediated by interactions of an adenine + uracil (AU)-rich element (ARE) in the 3′-UTR of bcl-2 mRNA with AU-binding proteins (AUBPs). Here, we identify and characterize ζ-crystallin as a new bcl-2 AUBP, whose silencing or overexpression has impact on bcl-2 mRNA stability. An increased Bcl-2 level observed in normal phytohemagglutinin (PHA)-activated T lymphocytes, acute lymphatic leukemia (ALL) T-cell lines, and T cells of patients with leukemia in comparison with normal non-PHA-activated T lymphocytes was concomitant with an increase in ζ-crystallin level. The specific association of ζ-crystallin with the bcl-2 ARE was significantly enhanced in T cells of patients with ALL, which accounts for the higher stability of bcl-2 mRNA and suggests a possible contribution of ζ-crystallin to bcl-2 overexpression occurring in this leukemia.—Lapucci, A., Lulli, M., Amedei, A., Papucci, L., Witort, E., Di Gesualdo, F., Bertolini, F., Brewer, G., Nicolin, A., Bevilacqua, A., Schiavone, N., Morello, D., Donnini, M., Capaccioli, S. ζ-Crystallin is a bcl-2 mRNA binding protein involved in bcl-2 overexpression in T-cell acute lymphocytic leukemia.

Akt activation assists tumor cell survival and promotes resistance to chemotherapy. Here we show that constitutively active Akt (CA-Akt) cells are highly sensitized to cell death induced by nutrient and growth factor deprivation, whereas dominant-negative Akt (DN-Akt) cells have a high rate of survival. The content of autophagosomes in starved CA-Akt cells was high, while DN-Akt cells expressed autophagic vacuoles constitutively, independently of nutrition conditions. Thus Akt down-regulation and downstream events can induce autophagosomes which were not directly determinants of cell death. Biochemical analysis in Akt-mutated cells show that (i) Akt and mTOR proteins were degraded more rapidly than the housekeeping proteins, (ii) mTOR phosphorylation at position Thr(2446) was relatively high in DN-Akt and low in CA-Akt cells, induced by starvation in mock cells only, which suggests reduced autoregulation of these pathways in Akt-mutated cells, (iii) both protein synthesis and protein degradation were significantly higher in starved CA-Akt cells than in starved DN-Akt cells or mock cells. In conclusion, constitutively active Akt, unable to control synthesis and wasting of proteins, accelerates the death of starved cells.

BACKGROUND In prostate cancer, mutations of the phosphatase PTEN can activate the kinase cascade PI3K/Akt/mTOR which induces drug resistance. METHODS Chemosensitization by siRNA targeting Akt was studied in HEK293 cells forced to express CA-Akt or kinase-dead DN-Akt. To decrease drug resistance, Akt was silenced with siRNA in human prostate DU-145 cell line expressing the normal PTEN or in LNCaP and PC3 cell lines expressing mutated-PTEN. Taxol was used for the chemosensitization studies. RESULTS Silencing Akt in the drug-resistant CA-Akt cells efficiently sensitized cells to antitubule agents, whereas silencing drug-responsive DN-Akt cells did not. Only minor effects were obtained in wild-type HEK293 cells. Potentiation by siRNA of taxol cytotoxicity was significantly greater in mutated-PTEN cells than in prostate cells expressing wild-type PTEN. The apoptotic program induced by taxol was preferentially potentiated by Akt siRNA in PTEN-mutated cell lines as regards the DU-145 cell line. CONCLUSIONS Silencing Akt in PTEN-mutated prostate cancer cells enhances the antitumor effects of taxol. No siRNA chemosensitization was obtained in prostate cells with wild type PTEN. Prostate 67: 782–789, 2007. © 2007 Wiley-Liss, Inc.

RNA has become a promising target for pharmacological purposes. Most current strategies are directed toward down-regulating its functions. In this study, we provide evidence of the up-regulation of messenger RNA in a sequence-specific manner. The bcl2 (b)-ARE (adenine-uridine-rich element) in the 3'-untranslated region of the b-RNA that regulates the rate of RNA degradation has been targeted with three chemically modified oligoribonucleotides designed in the antisense orientation (asORNs). The three asORNs were studied by a cell-free degradation assay. All three slowed the rate of RNA decay in a dose-response fashion, they were specific to the b-ARE, and two of them were individually effective. asORNs were then transfected into the malignant cells in culture and b-RNA half-life was measured by real-time reverse transcriptase-polymerase chain reaction. We showed that by stabilizing b-RNA the three asORNs increased the expression of b-RNA and of the relevant protein in a dose-response fashion.

Modulation of mRNA stability by regulatory cis-acting AU-rich elements (AREs) and ARE-binding proteins is an important posttranscriptional mechanism of gene expression control. We previously demonstrated that the 3′-untranslated region of BCL-2 mRNA contains an ARE that accounts for rapid BCL-2 down-regulation in response to apoptotic stimuli. We also demonstrated that the BCL-2 ARE core interacts with a number of ARE-binding proteins, one of which is AU-rich factor 1/heterogeneous nuclear ribonucleoprotein D, known for its interaction with mRNA elements of others genes. In an attempt to search for other BCL-2 mRNA-binding proteins, we used the yeast RNA three-hybrid system assay and identified a novel human protein that interacts with BCL-2 ARE. We refer to it as TINO. The predicted protein sequence of TINO reveals two amino-terminal heterogeneous nuclear ribonucleoprotein K homology motifs for nucleic acid binding and a carboxyl-terminal RING domain, endowed with a putative E3 ubiquitin-protein ligase activity. In addition the novel protein is evolutionarily conserved; the two following orthologous proteins have been identified with protein-protein BLAST: posterior end mark-3 (PEM-3) of Ciona savignyi and muscle excess protein-3 (MEX-3) of Caenorhabditis elegans. Upon binding, TINO destabilizes a chimeric reporter construct containing the BCL-2 ARE sequence, revealing a negative regulatory action on BCL-2 gene expression at the posttranscriptional level.

Maneuvering single gene expression is not only an optimal way to study gene function but also an ambitious goal, which will lead to the treatment of a variety of human diseases whose main pathogenetic event is a genetic alteration. The recent efforts focusing on the genome project have led to array based, high throughput, gene expression analysis techniques that allow the study of complex molecular networks. Combining these powerful new technologies with modulation of gene expressions is making it possible to unravel complex molecular networks or, vice versa, to find new gene products responsible for pathological conditions on which exogenous modulation can be productive. Efficient and specific modulation of gene expression can be obtained either by producing transgenic or gene knockout organisms or cells (gene targeting), or by treating organisms or cells with short synthetic nucleic acid segments in antisense orientation with respect to the targeted mRNAs (mRNA targeting by antisense strategy). While genome manipulation is a time consuming and expensive approach, requiring invasive intervention, the "antisense strategy" is characterized by high flexibility resulting from safeness, specificity, reversibility, modulability, and low cost. The rationale of the antisense strategy is that, once one gene sequence is known, its expression can be silenced by application of synthetic single-strand nucleic acid segments (oligonucleotides) whose sequence is in antisense orientation compared to the targeted mRNA. Recently, this "informational" strategy has been boosted by the discovery of the RNA interference: a natural mechanism by which cells are thought to fight detrimental exogenous viruses and endogenous transposons. Despite promising futures, antisense-based therapeutics are far from being an established reality. This review analyses the recent improvements in antisense-based gene expression modulation, focuses on the treatment of diseases in the light of the past, and provides our personal findings on this topic.

Recent evidence suggests that gene expression may be regulated, at least in part, at post-transcriptional level by factors inducing the extremely rapid degradation of messenger RNAs. These factors include reactions between adenyl-uridyl-rich elements (AREs) of the relevant mRNA and either specific proteins that bind to these elements or exosomes. This review deals with examples of the proteins (AU-rich binding proteins, AUBPs) and exosomes, which have been shown to form complexes with AREs and bring about rapid degradation of the relevant mRNA, and with certain other factors, which protect the RNA from such degradation. The biochemical and physiological factors underlying the stability of messenger RNAs carrying the ARE motifs will be reviewed in the light of their emerging significance for cell physiology, human pathology, and molecular medicine. We also consider the possible application of the results of recent insights into the mechanisms to pharmacological interventions to prevent or cure disorders, especially developmental disorders, which the suppression of gene expression may bring about. Molecular targeting of specific steps in protein degradation by synthetic compounds has already been utilized for the development of pharmacological therapies.

The expression of genes requiring finely tuned control is regulated by a posttranscriptional mechanism involving mRNA A + U-rich elements (AREs) cooperating with ARE-binding proteins (AUBPs) in modulation of mRNA stability. We reported previously that an ARE in the bcl-2 mRNA 3'-untranslated region (3'-UTR) had destabilizing activity and was involved in bcl-2 downregulation during apoptosis in vitro. Here we demonstrate that the bcl-2 ARE complexes with a number of specific AUBPs, whose pattern undergoes changes following application of apoptotic stimuli. The caspase inhibitor Z-VAD-fmk strongly attenuates both bcl-2 mRNA decay and bcl-2 AUBP pattern changes elicited by apoptotic stimuli, indicating the involvement of bcl-2 AUBPs in bcl-2 mRNA stability control.

In most human follicular B cell lymphomas the bcl-2 gene is up-regulated as a result of the t(14;18) chromosomal translocation generating a hybrid bcl-2–IgH mRNA. Recently, we have identified in t(14;18)-positive cells a bcl-2–IgH mRNA in the antisense orientation, putatively responsible for the overexpression of bcl-2 . Herein we show that this chimeric antisense transcript is an optimal target for synthetic oligodeoxynucleotides (ODNs). A variety of sense-oriented oligonucleotides have been designed that target the antisense transcript in regions endowed with a sequence specificity presumably restricted to an individual cell line (the bcl-2–IgH fusion regions) or extended to all t(14;18) cells (the ectopic bcl-2 segment upstream from the major breakpoint region and the IgH segment). All sense-oriented ODNs complementary to the antisense transcript induced an early strong inhibition of cell growth and a late fulminant cell death. As expected, the activity of ODNs targeting the fusion region was restricted to each individual cell line, whereas the activity of all ODNs targeting the common bcl-2 and IgH segments was extended to all t(14;18) cell lines tested. These sense ODNs were not effective in untranslocated cell lines. Antisense-oriented ODNs, complementary to the bcl-2–IgH mRNA, and control ODNs (scrambled, inverted, or mismatched) were biologically ineffective. The selectivity and efficacy of all sense ODNs tested provide support for the development of therapeutic ODNs targeting the bcl-2–IgH antisense transcript expressed in human follicular lymphomas.

An oligodeoxynucleotide (ODN), capable of reducing the growth of human B lymphocytes carrying the t(14;18) chromosome translocation, was prepared in different 'chemical versions': unmodified phosphodiester, phosphorothioate and phosphodiester capped with L-2'-deoxycytidine. Their binding affinity to the complementary synthetic target was studied by the melting point assay. The ODNs, administered to DOHH2 cells, were compared for stability in the culture medium, cellular uptake, time course of the intact sequence concentration within the cell and ability to inhibit cell growth. The 5', 3'-L-capped derivative and the phosphorothioate had comparable potency, superior to that of the unmodified ODN, in agreement with the concentration of undegraded ODNs within the cell.

Three techniques are evaluated for assessing the purity of synthetic oligodeoxyribonucleotides: reversed phase high-performance liquid chromatography (RP-HPLC), polyacrylamide gel-slab electrophoresis (PAGE), and capillary zone electrophoresis highly concentrated (18% T) entangled polymer networks (CZE). RP-HPLC does not seem to be able to discriminate and resolve the spectrum of failed sequences expected to accompany an oligonucleotide of a given length. The purity data, as given by the manufacturer, are most often close to 100%. PAGE in 20% T matrices, followed by ethidium bromide staining, gives a good resolution of failure sequences and purity assessments decidedly more realistic. CZE in 18% liquid polyacrylamide is able to resolve to baseline all shorter fragments and to give a precise evaluation of the amount of impurities, based on the intrinsic DNA absorbance at 254 nm. Most of the 18 mer oligonucleotides (and of their phosphorothioate derivatives) analyzed by us, as supplied by three different manufacturers, were found to be contaminated by a spectrum of failed and truncated sequences, ranging in size from 7 mer to 17 mer. The purity data rarely exceeded 80% and most often were of the order of 60%-70%. Conditions for a good routine performance of the CZE technique are described.

The antiproliferative effects mediated by a 14-mer homopyrimidine oligonucleotide (5' CTTTCT-CTTTTCTC3'), designed to form DNA triplex with a purine region of the DNA polymerase alpha promoter, were evaluated on the human breast cancer cell line MDA-MB 231. In order to stabilize the triple complex under physiologic conditions, replacement of cytosines by methylcytosines in the oligomer sequence was carried out. Band-shift analyses demonstrated a complete triplex formation between the radiolabeled target duplex DNA and the methylcytosine-modified oligomer at the concentration of 0.1 microM under physiologic pH and temperature. A single exposure of MDA-MB 231 cells to 0.5 microM methylcytosine-modified oligonucleotide was able to markedly reduce the cell number and the percentage of cells in DNA synthesis up to 58% and 66%, respectively, compared with controls. Furthermore, a 48% reduction in the amount of the DNA polymerase alpha mRNA was reported after treatment with the oligomer. In conclusion, data from the present study demonstrate that an oligonucleotide to DNA polymerase alpha promoter, designed to form a triple helix with target double-stranded DNA, inhibits the expression of the reporter gene at the biologic and molecular levels, suggesting a possible triplex-mediated mechanism of action.

Antisense oligonucleotides appear to offer considerable promise as sequence-specific inhibitors of gene expression. Different cellular targets for oligodeoxynucleotides with oncologic interest have been identified such as oncogenes, growth factors, and cell cycle-related genes. DNA polymerase alpha (pol alpha) plays a relevant role in DNA synthesis and cell proliferation. Pol alpha gene expression is constitutive throughout the cell cycle and its mRNA content and activity are related to the growth rate and neoplastic phenotype. The effects of a 18-mer pol alpha antisense oligomer on the proliferation of the MDA-MB 231 breast cancer cell line have been investigated. After 48 h in culture with oligomers (10 microM), about 50% growth inhibition was observed in antisense-treated cells, as evaluated by 3-(4,5-dimethythiazol-2yl)-2,5-diphenyltetrazolium bromide assay and cell count. [3H]Thymidine incorporation exhibited a 90% inhibition of DNA synthesis associated to 64% accumulation of cells at the G1-S border of the cycle as by flow cytometry, at 24 h. Northern hybridization and SDS-PAGE of immunoprecipitated MDA-MB 231 cell lysates revealed a decreased expression of pol alpha mRNA and a reduction of the 180-kDa polypeptide, respectively. Collectively, the data further confirm the relevance of pol alpha in the replicative cycle, as well as strengthen the potentiality of the antisense strategy for the control of gene expression and cell growth.

A subline of the human B cell lymphoma DHL-4, grown in the artificial serum-free medium HB101, displayed a resistant phenotype to the activity of an antisense oligodeoxynucleotide (aODN) effective on the parental DHL-4 line. It was found that the cellular uptake of the 18mer aODN in the two cell lines was almost the same. In contrast, the unresponsive subline DHL-4r degraded the aODN very efficiently, in contrast to the stability of aODN inside cells of the parental DHL-4 line. Activation of the degrading 'machinery' combined with selective properties of the artificial medium may be responsible for the loss of responsiveness to aODN.

Photodynamic therapy (PDT) is a treatment for cancer based on the photosensitization of tumor cells by photosensitive drugs and their subsequent destruction on exposure to light of particular wavelength. The combination of drug uptake in malignant tissues and selective delivery of laser-generated light provides an effective therapy with efficient tumor citotoxicity and minimal normal tissue damage. Since immune response of the host is important in the control of tumor growth and spreading, PDT is able to increase the antitumor immunity. In our laboratory we examined the antitumor effect of combination of PDT, with photoactivated M-THPC (meta-tetrahydroxyphenylchlorin, FOSCAN, Temoporphirin), adoptive immunotherapy, with immune lymphocytes, and chemotherapy on advanced murine tumors. Mice bearing L1210 tumor were treated at day +4 with Navelbine (NVB 1mg/Kg), at day +5,+6 with PDT (0.3mg/Kg of mTHPC and 100mW/cm(2) x 200'' of exposure of laser light), and at day + 7 with immune lymphocytes(IL), collected from mice pretreated with PDT(2x10(7) cells). The results show that the combination NVB + PDT + IL demonstrates a significant synergistic antitumor effect while the chemotherapy treatment with low dose of the drug is uneffective. The same positive results were obtained with the combination of Cisplatin (CDDP 0.5mg/Kg), PDT and IL, while the CDDP treatment alone is completely uneffective. In conclusion, these results suggest that it is possible to completely cure animals bearing advanced tumors, with a combined therapy, PDT + adoptive immunotherapy + low dose chemotherapy.

In the 3′-untranslated region, the destabilizing adenine-uridine (AU)-rich elements (AREs) control the expression of several transcripts through interactions with ARE-binding proteins (AUBPs) and RNA degradation machinery. Although the fundamental role for AUBPs and associated factors in eliciting ARE-dependent degradation of cognate mRNAs has been recently highlighted, the molecular mechanisms underlying the specific regulation of individual mRNA turnover have not yet been fully elucidated. Here we focused on the post-transcriptional regulation of bcl-2 mRNA in human cell lines under different conditions and genetic backgrounds. In the context of an AUBPs silencing approach, HuR knockdown reduced the expression of endogenous bcl-2, whereas unexpectedly, a bcl-2 ARE-reporter transcript increased significantly, suggesting that HuR expression has opposite effects on endogenous and ectopic bcl-2 ARE. Moreover, evidence was provided for the essential, specific and dose-dependent role of the Bcl-2 protein in regulating the decay kinetics of its own mRNA, as ascertained by a luciferase reporter system. Altogether, the data support a model whereby the Bcl-2 protein is the major determinant of its own ARE-dependent transcript half-life in living cells and its effect overcomes the activity of ARE-binding proteins.

Within three repeated 7-day incubation periods with either methotrexate (MTX) or trimetrexate (TMTX), human colon adenocarcinoma cells (HCT-8) developed high levels of resistance to these drugs, as evidenced by approximately 20- and 50-fold increases, respectively, in the median effective doses. Similarly, within six short-term exposures (4 hours) to the same drugs, a high degree of resistance developed in the cells. Alternating 4-hour treatment cycles with MTX and TMTX did not delay the onset of resistance to these antimetabolites in the HCT-8 cells. The same strategy produced no better results than giving either MTX or TMTX alone to (C57BL/6 x DBA/2)F1 mice bearing murine leukemia P388 cells. Furthermore, HCT-8 cells resistant to short-term (4-hour) exposure to MTX were cross-resistant to the same drug given for 7 days continuously, and cells resistant to MTX given continuously for 7 days were cross-resistant to the same drug given for 4 hours. Analogous results were obtained with TMTX, indicating that, under these circumstances, changing the schedule of administration of the same agent does not overcome resistance to it. The clinical relevance of these data to prolonged adjuvant chemotherapy, as well as loco-regional and continuous-infusion chemotherapy, is discussed.

We have identified previously a destabilizing adenine- and uracil-rich element (ARE) in the 3'-UTR of bcl-2 mRNA that interacted with ARE-binding proteins to down-regulate bcl-2 gene expression in response to apoptotic stimuli. We have also described three contiguous 2'-O-methyl oligoribonucleotides (ORNs) in both sense and antisense orientation with respect to the bcl-2 ARE that are able to regulate the bcl-2 mRNA half-life and Bcl-2 protein level in two different cell lines. Here we show that treatment of neuronal cell line (SHSY-5Y) with antisense ORNs targeting the bcl-2 ARE (bcl-2 ARE asORNs) prevents bcl-2 down-regulation in response to apoptotic stimuli with glucose/growth factor starvation (Locke medium) or oxygen deprivation and enhances the apoptotic threshold as evaluated by time-lapse videomicroscopy, fluorescence-activated cell sorting analysis, and caspase-3 activation. Additional effects of bcl-2 ARE asORNs included inhibition of cell cycle entry and a marked increase of cellular neurite number and length, a hallmark of neuronal differentiation resulting from bcl-2 up-regulation. The ability of bcl-2 ARE asORNs to enhance the apoptotic threshold and to induce neuronal differentiation implies their potential application as a novel informational tool to protect cells from ischemic damage and to prevent neuronal degeneration.

The in vitro efficacy of both EGFR inhibitor gefitinib and mTor inhibitor rapamycin, either administrated alone or in different combination schedules, was analysed in four pancreas cancer cell lines. Both drugs were found to induce cell growth inhibition, apoptosis as well as a slight but stable accumulation of cells in the G0/G1 phase. In all cell lines, neither gefitinib nor rapamycin affected EGFR and the expression of its downstream effectors. By contrast, gefitinib inhibited in a fast and completely way p-EGFR and partially p-Akt while a 3 days-rapamycin exposure resulted in the inhibition of the expression of both mTor and p70S6K. Moreover, after early stimulation, the mTor inhibitor produced a progressive, and almost complete inhibition of p-Akt. The analysis of combined gefitinib and rapamycin administration showed a clear schedule-dependent activity which turned out to be synergic only when gefitinib was given before rapamycin. This synergism seemed to depend on increase of both p-Akt and p70S6K inhibition, the greater the induction of apoptosis, the higher the decrease in cell cycle rate. Moreover, the antiangiogenic activity of the two drugs given in combination was demonstrated by a strong reduction of VEGF release which turned out to be more pronounced in the synergic schedule, and HIF-1α inhibition-independent. Our results suggest that the schedule of gefitinib followed by rapamycin, acting at different levels of the EGFR cellular pathway, could induce antitumor and antiangiogenic effects of clinical interest in the pancreas cancer model.

Adenine-uridine rich elements (AREs) play an important role in modulating mRNA stability, being the target site of many ARE-binding proteins (AUBPs) that are involved in the decay process. Three 26-mer 2'-O-methyl oligoribonucleotides (ORNs) homologous to the core region of ARE of bcl2 mRNA have been studied for decoy-aptamer activity in UV cross-linking assays. Sense-oriented ORNs competed with the ARE motif for the interaction with both destabilizing and stabilizing AUBPs in cell-free systems and in cell lines. Moreover, ORNs induced mRNA stabilization and up-regulated both Bcl2 mRNA and protein levels in the cells. Bcl2 ORNs stabilized other ARE-containing transcripts and up-regulated their expression. These results indicate that Bcl2 ORNs compete for AUBP-ARE interactions independently of ARE class and suggest that in the cell, the default labile status of ARE-containing mRNAs depends on the combined interaction of such transcripts with destabilizing AUBPs.

The mammalian target of rapamycin (mTOR) is a central regulator of ribosome biogenesis, protein synthesis, cell growth and neurite plasticity. The mTOR kinase controls the translation machinery, in response to amino acids and growth factors, via activation of p70 ribosomal S6 kinase (p70S6K) and inhibition of eIF-4E binding protein (4E-BP1). The mTOR protein belongs to the PI3K pathway activated by insulin, nutrients and growth factors. The PI3K pathway involves the Akt kinase, an upstream regulator of mTOR. Rapamycin is a potent immunosuppressant and investigational anticancer drug, which inhibits mTOR, blocking protein synthesis and arresting the cell cycle in G1 phase. A wide body of evidence supports the role of mTOR in cell signaling related to cell growth and proliferation. Nevertheless, our recent findings have revealed that mTOR may be also involved in a signaling pathway activated by microtubule-damaging drugs, including taxol and nocodazole. It is known that agents affecting the integrity of microtubules activate apoptotic program by inducing phosphorylation and inactivation of the antiapoptotic Bcl-2 protein in G2-M phase. We have some evidence that mTOR is involved in the enzymatic cascade that, starting from damaged microtubules, induces downstream phosphorylation of the Bcl-2 protein. We also found that the level of activity of Akt can regulate Bcl-2 phosphorylation, through the mTOR kinase. Since mTOR activation by survival signals occurs in G1 phase and damaged microtubules activate proapoptotic signals in G2-M phase, we suggest that mTOR might mediate these two different pathways in two different phases of the cell cycle.

Modulation of mRNA stability by regulatory cis-acting AU-rich elements (AREs) and ARE-binding proteins is an important posttranscriptional mechanism of gene expression control. We previously demonstrated that the 3'-untranslated region of BCL-2 mRNA contains an ARE that accounts for rapid BCL-2 down-regulation in response to apoptotic stimuli. We also demonstrated that the BCL-2 ARE core interacts with a number of ARE-binding proteins, one of which is AU-rich factor 1/heterogeneous nuclear ribonucleoprotein D, known for its interaction with mRNA elements of others genes. In an attempt to search for other BCL-2 mRNA-binding proteins, we used the yeast RNA three-hybrid system assay and identified a novel human protein that interacts with BCL-2 ARE. We refer to it as TINO. The predicted protein sequence of TINO reveals two amino-terminal heterogeneous nuclear ribonucleoprotein K homology motifs for nucleic acid binding and a carboxyl-terminal RING domain, endowed with a putative E3 ubiquitin-protein ligase activity. In addition the novel protein is evolutionarily conserved; the two following orthologous proteins have been identified with protein-protein BLAST: posterior end mark-3 (PEM-3) of Ciona savignyi and muscle excess protein-3 (MEX-3) of Caenorhabditis elegans. Upon binding, TINO destabilizes a chimeric reporter construct containing the BCL-2 ARE sequence, revealing a negative regulatory action on BCL-2 gene expression at the posttranscriptional level.

The serine/threonine kinase mTOR, the major sensor of cell growth along the PI3K/Akt pathway, can be activated by agents acting on microtubules. Damaged microtubules induce phosphorylation of the Bcl-2 protein and lower the threshold of programmed cell death, both of which are inhibited by rapamycin. In HEK293 cells expressing Akt mutants, the level of Bcl-2 phosphorylation and the threshold of apoptosis induced by taxol or by nocodazole are significantly modified. In cells expressing dominant-negative Akt (DN-Akt), Bcl-2 phosphorylation and p70S6KThr421/Ser424 phosphorylation induced by taxol or nocodazole were significantly enhanced as compared to cells expressing constitutively active Akt (CA-Akt) and inhibited by rapamycin. Moreover, DN-Akt cells were more sensitive to antitubule agents than CA-Akt cells. In nocodazole-treated HEK293 cells sorted according to cell cycle, the p70S6KThr421/Ser424 phosphorylation was associated to the G2/M fraction. More relevant, nocodazole inhibited, in a dose-response manner, mTOR phosphorylation at Ser2448. This activity, potentiated in DN-Akt cells, was not detectable in CA-Akt cells. Our results suggest that death signals originating from damaged microtubules in G2/M can compete with G1 survival pathways at the level of mTOR. These findings have implications for cancer therapy and drug resistance.

We have shown previously that the decay of human bcl-2 mRNA is mediated by an adenine/uridine-rich element (ARE) located in the 3′-untranslated region. Here, we have utilized a non-radioactive cell-free mRNA decay system to investigate the biochemical and functional mechanisms regulating the ARE-dependent degradation of bcl-2 mRNA. Using RNA substrates, mutants, and competitors, we found that decay is specific and ARE-dependent, although maximized by the ARE-flanking regions. In unfractionated extracts from different cell types and in whole cells, the relative enzymatic activity was related to the amount of Bcl-2 protein expressed by the cells at steady state. The degradation activity was lost upon Bcl-2 depletion and was reconstituted by adding recombinant Bcl-2. Ineffective extracts from cells that constitutively do not express Bcl-2 acquire full degradation activity by adding recombinant Bcl-2 protein. We conclude that Bcl-2 is necessary to activate the degradation complex on the relevant RNA target.

Synthetic ribozymes are catalytic RNA molecules designed to inhibit gene expression by cleaving specific mRNA sequences. We investigated the potential of synthetic ribozymes to inhibit bcl-2 expression in apoptosis defective bcl-2 overexpressing tumors. A chemically stabilized hammerhead ribozyme has been targeted to the A+U-rich regulative element of bcl-2 mRNA that is involved in bcl-2 gene switch-off during apoptosis. The design of the ribozyme was based on the results of probing accessibility of the RNA target in cellular extracts with antisense DNA. The ribozyme was lipotransfected to a bcl-2 overexpressing human lymphoma cell line (Raji). The cellular uptake of this ribozyme resulted in a marked reduction of both bcl-2 mRNA and BCL-2 protein levels and dramatically increased cellular death by apoptosis. Our results suggest a potential therapeutic application of such ribozyme for the treatment of bcl-2 overexpressing tumors.

We previously identified a conserved A + U-rich element (ARE) in the 3'-untranslated region of bcl-2 mRNA. We have also recently demonstrated that the bcl-2 ARE interacts with a number of ARE-binding proteins (AUBPs) whose pattern changes during apoptosis in association with bcl-2 mRNA half-life reduction. Here we show that the AUBP AUF1 binds in vitro to bcl-2 mRNA. The results obtained in a yeast RNA three-hybrid system have demonstrated that the 1-257-amino acid portion of p37 AUF1 (conserved in all isoforms), containing the two RNA recognition motifs, also binds to the bcl-2 ARE in vivo. UVC irradiation-induced apoptosis results in an increase of AUF1. Inhibition of apoptosis by a general caspase inhibitor reduces this increase by 2-3-fold. These results indicate involvement of AUF1 in the ARE/AUBP-mediated modulation of bcl-2 mRNA decay during apoptosis.

The immunosuppressant rapamycin, an immunophilin-binding antibiotic, has been studied in follicular B-cell lymphoma lines that express the highest level of the BCL-2 protein. The growth rate of human follicular B-cell lymphoma lines was slowed more efficiently than that of other human B-cell lines or non-B-cell lines. This effect was dependent on the arrest of cells in the G(1) phase; the number of apoptotic cells was not increased. Rapamycin inhibited apoptosis or caspase activation induced by cytotoxic drugs, whereas caspase activation by doxorubicin was not inhibited. The increase in the cellular concentration of BCL-2 protein was related to its concentration in the steady state and was unrelated to the amount of bcl-2 mRNA. The increase of BCL-2 level in the cells rather than its level in the steady state may be important for drug resistance. The biochemical target of rapamycin, the mTOR kinase, may be a candidate sensitising agent for chemotherapy. This effect of rapamycin shows that G(1) arrest and protection from apoptosis are combined events susceptible to regulation by pharmacological means.

Rapamycin, a specific inhibitor of the serine/threonine mTOR kinase, markedly inhibited both cell growth and apoptosis in human B-cell lines. Besides arresting cells in G(1) by increasing p27(kip1), rapamycin tripled the cellular level of the BCL-2 protein. The activity was dose-dependent and specific for the p27(kip1) and BCL-2 proteins. Rapamycin did not affect bcl-2 mRNA although it increased cellular BCL-2 concentration by inhibiting phosphorylation, a mechanism initiating the decay process. To add new insight, we combined rapamycin treatment with treatment by taxol, which, by damaging microtubules, can phosphorylate BCL-2 and activate apoptosis. It was found that the mTOR kinase was activated in cells treated with taxol or with nocodazole although it was inhibited in cells pre-treated with rapamycin. BCL-2 phosphorylation, apoptosis and hyperdiploidy were also inhibited by rapamycin. In contrast, taxol-induced microtubule stabilization or metaphase synchronization were not inhibited by rapamycin. Taken together, these findings indicate that mTOR belongs to the enzymatic cascade that, starting from damaged microtubules, phosphorylates BCL-2. By regulating apoptosis, in addition to the control of a multitude of growth-related pathways, mTOR plays a nodal role in signaling G(1) and G(2)-M events.

In an effort to identify potential upregulators of bcl-2 activity in t(14;18) follicular B lymphoma cells, we detected a hybrid bcl-2/IgH RNA transcribed in antisense orientation. This antisense transcript may contribute to upregulation of bcl-2 expression in t(14;18) cells, overlapping AU-rich motifs present in the 3′-untranslated region of bcl-2 mRNA. We have studied the enzymatic efficiency of a ribozyme directed towards the bcl-2 AU-rich region in a cell-free system determining its kinetic parameters.

Photodynamic therapy (PDT) is based on the administration of tumor-localizing photosensitizers followed by light exposure of the tumor mass. The photocytotoxic effects are mainly caused by the generation of singlet oxygen. Recently, PDT has been proposed for use in combination with anticancer chemotherapy with a view to exploiting any additive antitumor effect. We investigated the effect of PDT with photoactivated aluminum disulfonated phthalocyanine (AlS2Pc) combined with the antiblastic drugs Adriamycin (ADR) and cisplatinum (CDDP) on murine tumors. Mice bearing L1210 leukemia and P388 lymphoma were treated with ADR or CDDP and subsequently treated with PDT. Low chemotherapy doses were ineffective, but the combination of antiblastic drugs + PDT had a significantly additive antitumor effect. In conclusion, with this combined therapy we were able to greatly reduce the effective doses of antiblastic drugs, thus lowering their toxic effects on normal host tissues.

The effect of an antisense oligonucleotide to the K+ channel coding mKv1.1 mRNA on antinociception induced by the tricyclic antidepressants, clomipramine (20–35 mg kg−1 s.c.) and amitriptyline (10–25 mg kg−1 s.c.), was investigated in the mouse hot-plate test. Antisense oligonucleotide (0.5-1.0-2.0-3.0 nmol per i.c.v. injection) produced a dose-dependent inhibition of clomipramine and amitriptyline antinociception 72 h after the last i.c.v. injection. The sensitivity to both analgesic drugs returned 7 days after antisense oligonucleotide injection, indicating the absence of irreversible damage or toxicity. Treatment with a degenerated oligonucleotide did not modify the clomipramine- and amitriptyline-induced antinociception in comparison with that in naive (unpretreated controls), vector and saline i.c.v.-injected mice. A quantitative reverse transcription-polymerase chain reaction (RT-PCR) study demonstrated a reduction in mRNA levels only in the antisense oligonucleotide treated group. Antisense oligonucleotide, degenerated oligonucleotide or vector pretreatment, in the range of doses used, did not produce any behavioural impairment as revealed by the mouse rotarod and hole-board tests. The present results indicate that modulation of the mKv1.1 K+ channel plays an important role in the central analgesia induced by the tricyclic antidepressants, clomipramine and amitriptyline.

Photodynamic therapy (PDT) is a relatively new cancer treatment modality that employs light excitation of a photosensitizer to yield cytotoxic oxygen-related species. In the present study we explored whether PDT would have therapeutic effect against doxorubicin-resistant murine tumors. We compared the efficacy of PDT with aluminium disulphonated phthalocyanine (A1S2Pc) and laser light on the doxorubicin-sensitive murine tumors, B16 melanoma (B16), L1210 leukemia (L1210), P388 lymphoma (P388) and the corresponding doxorubicin-resistant lines (B16/Dx, L1210/Dx and P388/Dx). Mice bearing L1210-L1210/Dx, P388-P388/Dx and B16-B16/Dx, were treated with 5 mg/kg of A1S2Pc and laser light (100 mW/cm2 x 10 min of exposure) or with doxorubicin (10 or 12 mg/kg i.v.). The results show that PDT is active versus all tumors while doxorubicin is effective only against the three sensitive tumor lines (L1210, P388 and B16). These observations suggest that PDT might be a beneficial alternative treatment for drug-resistant tumors.

Since immunity is important in the control of tumor growth and spreading, in our laboratory we examine the effect of photoactivated phthalocyanines on the immune compartment. Spleen hyperplasia as well as spleen and marrow hypercellularities were observed in mice bearing MS-2 fibrosarcoma treated at different times with Aluminum disulfonated Phthalocyanine and laser light. Results suggest a potential `tumor immunity' induced by PDT with photoactivated phthalocyanines and that the dyes used in PDT could play an immunopharmacological role in cancer treatment.

Antisense oligonucleotides and their derivatives have been shown to be specific inhibitors of gene expression. They are considered a very promising new generation of drugs, potentially useful in most human diseases, including cancers and viral infections. The elegance of the antisense oligonucleotides lies in their ability to bind, via standard Watson-Crick base pairing, a complementary region within a target mRNA. Although easily synthesized, therapeutic applications have been restricted by a number of difficulties including: stability, pharmacokinetic behavior (both a cellular and at systemic level), and the high cost of industrial production. The object of this review is to briefly describe the major properties of antisense oligonucleotides, the modalities currently under investigation to circumvent the difficulties in their use, and the up-to-date experimental applications, including findings from our own laboratory. As very few oligonucleotides need to be synthesized in order to obtain an active compound, compared with an average of 10,000 new standard compounds, prospects are extremely exciting and worthy of maximum attention.

A 2780 human ovarian cancer cells, obtained from an untreated patient, have been exposed to a relatively low, clinically maintainable dose (10 nmol/l) of the anthracycline doxorubicin (DX) to derive a low-degree (5-fold) drug-resistant subline (A2780-DX1). Compared to parental cells, these DX-resistant cells have increased size (+60% of cell volume) and contain a greater number of cytoplasmic vacuoles as determined by electron microscopy. When exposed to several other antiproliferative drugs, A2780-DX1 cells were highly cross-resistant (greater than 10-fold) to epirubicin, mafosfamide and cisplatin and slightly cross-resistant (2- to 3-fold) to navelbine and bleomycin, while they retained the original sensitivity to vinblastine, Ara-C and fluorouracil. Gel electrophoresis of cytoplasmic membrane proteins showed differences between the pattern of parental A2780 sensitive and A2780-DX1 cells as far as low-molecular-weight proteins (less than 45 kD) are concerned, while no clear overexpression of P-glycoprotein (P-170) could be detected. Membrane modifications yielding a decrease of both DX uptake and retention, increased content of intracellular glutathione (+32%) and reduced DNA double-strand breaks seem to be involved in the resulting multidrug-resistant phenotype of A2780-DX1 cells.

To assess the prognostic value of tumor proliferative activity, 89 patients with operable non-small cell lung cancer were studied. Tumor samples were obtained during surgery and cell kinetics were analyzed by the in vitro thymidine labelling index (TLI). The overall median TLI (2.9) was used to identify two subsets of patients with high and low proliferating tumors. In univariate analysis survival was significantly longer in patients with lower TLI (P = 0.047) and with stage I-II (P = 0.003) and T1-T2 tumors (P = 0.043). In multivariate analysis, stage was the most important prognostic parameter (P = 0.004). The risk of death for patients with TLI higher than 2.9 was increased (hazard ratio = 2.01, CI = 0.96-4.27).

SUMMARY The experiments reported here concern the charcterization by techniques of in vitro cell-mediated immunity of the antigens induced by 5-(3,3'dimethyl-1-triazine)-imidazole-4-carboxamide (DTIC) on L1210, a chemically-induced lymphoma of DBA/2 mice (H-2d). This series of experiments with the DTIC-treated L1210 tumour show the presence of an H-2D'-like antigen which resembles the Dk gene product/s of the H-2k haplotype.

Immunologic alteration of the L5178Y lymphoma was obtained in vivo after treatment with 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DIC). A single dose of 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU) "CURED" MICE CHALLENGED WITH L5178Y cells that had been treated with DIC (L5178Y/DIC) for four transplant generations; BCNU did not cure mice bearing the parent tumor. The L5178Y/DIC, treated in vivo for five transplant generations, id not grow in syngeneic mice. L5178Y/OIC cell growth and incidences of death were similar to those of parent cells when inoculated into heavily immunosuppressed mice. Adoptive transfer of lymphocytes from spleens of mice sensitized to the drug-altered tumor specifically protected immunosuppressed mice bearing the L5178Y/DIC tumor. Little protection was afforded by lymphocytes immune to the parent L5178Y tumor, whereas nonimmune lymphocytes or lymphocytes immune against unrelated tumors were completely ineffective. Anti-L5178Y/DIC lymphocytes did not cure mice challenged with the parent L5178Y tumor. Irradiated (400 R) mice previously sensitized to L5178Y/DIC cells rejected 10(2)-10(7) inocula of L5178Y/DIC cells and died when the parent L5178Y was used for challenge. It was concluded that antigeni( alterations of L5178Y cells occurred in (BALB/ctcr X DBA/2Cr)F1 mice after treatment with DIC in vivo.

A hybridoma cell line (5F) secreting monoclonal antibodies directed to αDNA polymerase has been developed. Kinetic studies on peripheral blood lymphocytes stimulated with mitogen and human colon cancer cell lines established in vitro were made by the two autoradiographic techniques of Thymidine Labelling Inde and Primer-dependent αDNA polymerase Labelling Index and the immunoperoxidase assay (PAP) with monoclonal antibody to αDNA polymerase. We demonstrated the exclusively intranuclear presence of αDNA polymerase in lymphocytes induced to proliferate and actively growing colon cancer cells in contrast with the cytoplasmic distribution of the enzyme in resting stage populations. The feasibility of using monoclonal antibodies to αDNA polymerase to determine cell growth fraction was evaluated.

Summary Antigenic alterations of experimental lymphomas can be induced by a proper treatment with antineoplastic agents. While immunologic properties of drug-treated tumors have received some attention, surface characteristic of altered cells have found scant interest. Reduced susceptibility to Con A agglutination of L1210/DTIC cells and EL4/DTIC cells in respect to L1210 and EL4 parental cells has been obtained. Following adsorbing assays and agglutination inhibition assays by the sugar moiety αMM, similar amount of Con A has been bound by either original or by DTIC-treated cells. Con A binding sites on cell membranes of experimental lymphomas have not been modified by the DTIC treatment, however the mobility of surface molecules is likely to be altered.

Hematoporphyrin derivative (Hpd) is a fluorescent dye that is preferentially incorporated by tissues with a high mitotic index, such as tumor cells and blast cells. A cytotoxic effect is produced following light activation. Previous studies have shown a long lasting reversible inhibition of DNA synthesis in Hpd-treated cells that failed to stimulate allogeneic lymphocytes in either primary or in secondary MLR. In this study we report Hpd inhibitory effects on some immunological assays in vitro. Treatment with Hpd of cytotoxic effector cells resulted in inhibition of their lytic activity likely dependent on the loss of binding to target cells. In the same way Hpd treatment inactivated the lytic activity of NK cells. In contrast Hpd-treatment of target cells did not modify the above immunological reactions. Moreover Con A agglutinability, antibody dependent capping as well as E-rosettes were inhibited following an Hpd treatment of relevant cells. Since normal susceptibility to humoral and cell mediated lysis was exhibited by Hpd-treated cells it is unlikely that cell surface molecules were damaged. An inhibitory effect exerted by an Hpd treatment on cell surface movements might explain these findings.

Summary Concepts actually relevant to the experimental chemoimmunotherapy of cancer have been in short reviewed. Attempts to interperse effective chemotherapy and aspecific host immune stimulation or active immunotherapy by the use of in vitro altered tumor cells have been discussed. Increasing tumor cell immunogenicity by pharmacological means leads us to exploit original contributions to experimental chemoimmuno therapy of cancer.